Compositions and methods for performing hybridizations with separate denaturation of the sample and probe

ABSTRACT

The invention provides methods and compositions for separately denaturing a probe and target in hybridization applications. The invention may, for example, eliminate the use of, or reduce the dependence on formamide in hybridization applications. Compositions for use in the invention include an aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature double-stranded nucleotide sequences.

FIELD OF THE INVENTION

The present invention relates to compositions and methods forhybridization applications involving separate denaturation of the targetand probe. In one embodiment, the present invention can be used for thein vivo, in vitro, and in situ molecular examination of DNA and RNA. Inparticular, the invention can be used for the molecular examination ofDNA and RNA in the fields of cytology, histology, and molecular biology.In other embodiments, the present invention can be used for in situhybridization (ISH) applications.

BACKGROUND AND DESCRIPTION

Double stranded nucleic acid molecules (i.e., DNA (deoxyribonucleicacid), DNA/RNA (ribonucleic acid) and RNA/RNA) associate in a doublehelical configuration. This double helix structure is stabilized byhydrogen bonding between bases on opposite strands when bases are pairedin a particular way (A+T/U or G+C) and hydrophobic bonding among thestacked bases. Complementary base paring (hybridization) is central toall processes involving nucleic acid.

In a basic example of hybridization, nucleic acid probes or primers aredesigned to bind, or “hybridize,” with a target nucleic acid, forexample, DNA or RNA in a sample. One type of hybridization application,in situ hybridization (ISH), includes hybridization to a target in aspecimen wherein the specimen may be in vivo, in situ, or in vitro, forexample, fixed or adhered to a glass slide. The probes may be labeled tomake identification of the probe-target hybrid possible by use of afluorescence or bright field microscope/scanner.

The efficiency and accuracy of nucleic acid hybridization assays mostlydepend on at least one of three major factors: a) denaturationconditions, b) renaturation conditions, and c) post-hybridizationwashing conditions.

In order for probes or primers to bind to a target nucleic acid in asample, complementary strands of nucleic acid must be separated. Thisstrand separation step, termed “denaturation,” typically requiresaggressive conditions to disrupt the hydrogen and hydrophobic bonds inthe double helix. The probe and target molecules can either be denaturedseparately or together (co-denaturation). It has been argued thatseparate denaturation preserves morphology better, whereasco-denaturation reduces the number of practical steps. For thesereasons, separate denaturation steps are most often used in molecularcytogenetics applications, and co-denaturation is most often used whentissue sections are analyzed.

Traditional hybridization experiments, such as ISH assays, use aformamide-containing solution to denature doubled stranded nucleic acid.Formamide disrupts base pairing by displacing loosely and uniformlybound hydrate molecules, and by causing “formamidation” of theWatson-Crick binding sites. Thus, formamide has a destabilizing effecton double stranded nucleic acids and analogs.

Once the complementary strands of nucleic acid have been separated, a“renaturation” or “reannealing” step allows the primers or probes tobind to the target nucleic acid in the sample. This step is alsosometimes referred to as the “hybridization” step. Although formamidepromotes denaturation of double stranded nucleic acids and analogs, italso significantly prolongs the renaturation time, as compared toaqueous denaturation solutions without formamide. Indeed, there-annealing step is by far the most time-consuming aspect oftraditional hybridization applications. Examples of traditionalhybridization times are shown in FIGS. 1 and 2.

In addition, formamide has disadvantages beyond a long processing time.Formamide is a toxic, hazardous material, and is subject to strictregulations for use and waste. Furthermore, the use of a highconcentration of formamide can cause morphological destruction ofcellular, nuclear, and/or chromosomal structure, resulting in highbackground signals during detection.

Thus, a need exists for overcoming the drawbacks associated with priorart hybridization applications. By addressing this need, the presentinvention provides several potential advantages over prior arthybridization applications.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods andcompositions which result in hybridization applications having at leastone of the following advantages over prior art hybridizationapplications: lower background, more homogenous background, preservationof sample morphology, easier automation, faster procedure, and safer(less-toxic) reagents. One way in which the present invention achievesthose objectives is by providing methods and compositions for separatedenaturation of the probe and the target.

The compositions and methods of the invention are applicable to anyhybridization technique. The compositions and methods of the inventionare also applicable to any molecular system that hybridizes or bindsusing base pairing, such as, for example, DNA, RNA, PNA, LNA, andsynthetic and natural analogs thereof.

The nucleic acid hybridization methods and compositions of the presentinvention may be used for the in vivo, in vitro, or in situ analysis ofgenomic DNA, chromosomes, chromosome fragments, genes, and chromosomeaberrations such as translocations, deletions, amplifications,insertions, mutations, or inversions associated with a normal conditionor a disease. Further, the methods and compositions are useful fordetection of infectious agents as well as changes in levels ofexpression of RNA, e.g., mRNA and its complementary DNA (cDNA).

Other uses include the in vivo, in vitro, or in situ analysis ofmessenger RNA (mRNA), viral RNA, viral DNA, small interfering RNA(siRNA), small nuclear RNA (snRNA), non-coding RNA (ncRNA, e.g., tRNAand rRNA), transfer messenger RNA (tmRNA), micro RNA (miRNA),piwi-interacting RNA (piRNA), long noncoding RNA, small nucleolar RNA(snoRNA), antisense RNA, double-stranded RNA (dsRNA), methylations andother base modifications, single nucleotide polymorphisms (SNPs), copynumber variations (CNVs), and nucleic acids labeled with, e.g.,radioisotopes, fluorescent molecules, biotin, digoxigenin (DIG), orantigens, alone or in combination with unlabeled nucleic acids.

The nucleic acid hybridization method and compositions of the presentinvention are useful for in vivo, in vitro, or in situ analysis ofnucleic acids using techniques such as northern blot, Southern blot,flow cytometry, autoradiography, fluorescence microscopy,chemiluminescence, immunohistochemistry, virtual karyotype, gene assay,DNA microarray (e.g., array comparative genomic hybridization (arrayCGH)), gene expression profiling, Gene ID, Tiling array, gelelectrophoresis, capillary electrophoresis, and in situ hybridizationssuch as FISH, SISH, CISH. The methods and compositions of the inventionmay be used on in vitro and in vivo samples such as bone marrow smears,blood smears, paraffin embedded tissue preparations, enzymaticallydissociated tissue samples, bone marrow, amniocytes, cytospinpreparations, imprints, etc.

In one embodiment, the invention provides methods and compositions forhybridizing at least one molecule (e.g., a probe) to a target (e.g., abiological sample) using separate denaturation steps for the moleculeand target. In other embodiments, the invention may eliminate the useof, or reduce the dependence on formamide in such denaturation stepsfrom, e.g., 70% formamide in traditional denaturation buffers to 50%,25%, 15%, 10%, 5%, 2%, 1% or 0% v/v formamide in the compositions andmethods of the invention. Thus, in some aspects, the present inventionovercomes several disadvantages associated with traditionalhybridization assays, including the major toxicity issue and the timeconsuming renaturation step associated with the use of formamide in suchtraditional hybridization assays.

One aspect of the invention is a composition or solution for separatelydenaturing a probe and target in hybridization applications. Thecomposition for denaturing the target may comprise the same componentsas the composition for denaturing the probe, or the two compositions maycomprise different components. Compositions for use in the invention mayinclude an aqueous composition comprising at least one polar aproticsolvent in an amount effective to denature double-stranded nucleotidesequences. An amount effective to denature double-stranded nucleotidesequences is an amount that enables hybridization. For example, one wayto test for whether the amount of polar aprotic solvent is effective toenable hybridization is to determine whether the polar aprotic solvent,when used in the hybridization methods and compositions describedherein, such as example 1, yield a detectable signal and/or an amplifiednucleic acid product.

Non-limiting examples of effective amounts of polar aprotic solventsinclude, e.g., about 1% to about 95% (v/v). In some embodiments, theconcentration of polar aprotic solvent is 5% to 60% (v/v). In otherembodiments, the concentration of polar aprotic solvent is 10% to 60%(v/v). In still other embodiments, the concentration of polar aproticsolvent is 30% to 50% (v/v). Concentrations of 1% to 5%, 5% to 10%, 10%,10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, or 60% to70% (v/v) are also suitable. In some embodiments, the polar aproticsolvent will be present at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%,3%, 4%, or 5% (v/v). In other embodiments, the polar aprotic solventwill be present at a concentration of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%,10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%,16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% (v/v).

According to another aspect of the present invention the aqueouscompositions comprising a polar aprotic solvent have reduced toxicity.For example, a less-toxic composition than traditional solutions used inhybridization applications may comprise a composition with the provisothat the composition does not contain formamide, or with the provisothat the composition contains less than 25%, or less than 10%, or lessthan 5%, or less than 2%, or less than 1%, or less than 0.5%, or lessthan 0.1%, or less than 0.05%, or less than 0.01% formamide. Aless-toxic composition may, in one embodiment, also comprise acomposition with the proviso that the composition does not containdimethyl sulfoxide (DMSO), or with the proviso that the compositioncontains less than 25%, 10%, 5%, 2%, or less than 1%, or less than 0.5%,or less than 0.1%, or less than 0.05%, or less than 0.01% DMSO.

In one aspect of the invention, suitable polar aprotic solvents for usein the invention may be selected based on their Hansen SolubilityParameters. For example, suitable polar aprotic solvents may have adispersion solubility parameter between 17.7 to 22.0 MPa^(1/2), a polarsolubility parameter between 13 to 23 MPa^(1/2), and a hydrogen bondingsolubility parameter between 3 to 13 MPa^(1/2).

According to one aspect of the present invention, suitable polar aproticsolvents for use in the invention are cyclic compounds. A cycliccompound has a cyclic base structure. Examples include the cycliccompounds disclosed herein. In other embodiments, the polar aproticsolvent may be chosen from Formulas 1-4 below:

where X is O and R1 is alkyldiyl.

According to another aspect of the invention, suitable polar aproticsolvents for use in the invention may be chosen from Formula 5 below:

where X is optional and if present, is chosen from O or S;

where Z is optional and if present, is chosen from O or S;

where A and B independently are O or N or S or part of the alkyldiyl ora primary amine;

where R is alkyldiyl; and

where Y is O or S or C.

Examples of suitable polar aprotic solvents according to Formula 5 areprovided in Formulas 6-9 below:

According to yet another aspect of the invention the polar aproticsolvent has lactone, sulfone, nitrile, sulfite, or carbonatefunctionality. Such compounds are distinguished by their relatively highdielectric constants, high dipole moments, and solubility in water.

According to another aspect of the invention the polar aprotic solventhaving lactone functionality is γ-butyrolactone (GBL), the polar aproticsolvent having sulfone functionality is sulfolane (SL), the polaraprotic solvent having nitrile functionality is acetonitrile (AN), thepolar aprotic solvent having sulfite functionality is glycolsulfite/ethylene sulfite (GS), and the polar aprotic solvent havingcarbonate functionality is ethylene carbonate (EC), propylene carbonate(PC), or ethylene thiocarbonate (ETC). In yet another aspect of theinvention, the compositions and methods of the invention comprise apolar aprotic solvent, with the proviso that the polar aprotic solventis not acetonitrile (AN) or sulfolane (SL).

According to yet another aspect, the invention discloses a method ofhybridizing nucleic acid sequences comprising:

-   -   combining a first nucleic acid sequence with a first aqueous        composition comprising at least one polar aprotic solvent in an        amount effective to denature a double-stranded nucleotide        sequence,    -   combining a second nucleic acid sequence with a second aqueous        composition comprising at least one denaturing agent in an        amount effective to denature double-stranded nucleotide        sequence, and    -   combining the first and the second nucleic acid sequences for at        least a time period sufficient to hybridize the first and second        nucleic acid sequences.

According to yet another aspect, the invention discloses a method ofhybridizing nucleic acid sequences comprising:

-   -   combining a first nucleic acid sequence with a first aqueous        composition comprising at least one polar aprotic solvent in an        amount effective to denature a double-stranded nucleotide        sequence, and    -   applying a second aqueous composition comprising a second        nucleic acid sequence and at least one denaturing agent in an        amount effective to denature double-stranded nucleotide        sequences to said first nucleic acid sequence for at least a        time period sufficient to hybridize the first and second nucleic        acid sequences.

In one embodiment, the denaturing agent in the second aqueouscomposition is a polar aprotic solvent. In one embodiment, the first andsecond aqueous compositions comprise the same components. In anotherembodiment, the first and second aqueous compositions comprise differentcomponents.

In one embodiment, the first nucleic acid sequence is in a biologicalsample. In another embodiment, the biological sample is a cytology orhistology sample.

In one embodiment, the first nucleic acid sequence is a single strandedsequence and the second nucleic acid sequence is a double strandedsequence. In another embodiment, the first nucleic acid sequence is adouble stranded sequence and the second nucleic acid sequence is asingle stranded sequence. In yet another embodiment, both the first andsecond nucleic acid sequences are double stranded. In yet anotherembodiment, both the first and second nucleic acid sequences are singlestranded.

In one embodiment, a sufficient amount of time to denature the firstnucleic acid sequence is provided. In one embodiment, a sufficientamount of energy to denature the first nucleic acid sequence isprovided. In another embodiment, a sufficient amount of time to denaturethe second nucleic acid sequence is provided. In another embodiment, asufficient amount of energy to denature the second nucleic acid sequenceis provided. In another embodiment, a sufficient amount of time tohybridize the first and second nucleic acid is provided. In anotherembodiment, a sufficient amount of energy to hybridize the first andsecond nucleic acids is provided.

According to yet another aspect of the present invention, the energy isprovided by heating the aqueous compositions and nucleic acid sequences.Thus, the method of the invention may include the steps of heating andcooling the aqueous compositions and nucleic acid sequences.

A further aspect of the invention comprises a method wherein the step ofproviding a sufficient amount of energy involves a heating stepperformed by the use of microwaves, hot baths, hot plates, heat wire,peltier element, induction heating, or heat lamps.

According to a further aspect, the invention relates to the use of acomposition comprising between 1 and 95% (v/v) of at least one polaraprotic solvent for the separate denaturation of a target and sample ina hybridization application.

According to yet another aspect, the invention relates to kitscomprising the compositions of the invention for use in hybridizationassays in which the probe and target are denatured separately.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a typical time-course for single locus detection withprimary labeled FISH probes co-denatured with formaldehyde fixedparaffin embedded tissue sections (histological specimens). The barsrepresent a hybridization assay performed using traditionalhybridization solutions. The first bar on the left represents thedeparaffination step; the second bar represents the heat-pretreatmentstep; the third bar represents the digestion step; the fourth barrepresents the denaturation and hybridization steps; the fifth barrepresents the stringency wash step; and the sixth bar represents themounting step.

FIG. 2 depicts a typical time-course for single locus detection withprimary labeled FISH probes co-denatured with cytological specimens. Thebars represent a hybridization assay performed using a traditionalhybridization solutions. The first bar on the left represents thefixation step; the second bar represents the denaturation andhybridization steps; the third bar represents the stringency wash step;and the fourth bar represents the mounting step.

DETAILED DESCRIPTION A. Definitions

In the context of the present invention the following terms are to beunderstood as follows:

“Biological sample” is to be understood as any in vivo, in vitro, or insitu sample of one or more cells or cell fragments. This can, forexample, be a unicellular or multicellular organism, tissue section,cytological sample, chromosome spread, purified nucleic acid sequences,artificially made nucleic acid sequences made by, e.g., a biologic basedsystem or by chemical synthesis, microarray, or other form of nucleicacid chip. In one embodiment, a sample is a mammalian sample, such as,e.g., a human, murine, rat, feline, or canine sample.

“Nucleic acid,” “nucleic acid chain,” and “nucleic acid sequence” meananything that binds or hybridizes using base pairing including,oligomers or polymers having a backbone formed from naturally occurringnucleotides and/or nucleic acid analogs comprising nonstandardnucleobases and/or nonstandard backbones (e.g., a peptide nucleic acid(PNA) or locked nucleic acid (LNA)), or any derivatized form of anucleic acid.

As used herein, the term “peptide nucleic acid” or “PNA” means asynthetic polymer having a polyamide backbone with pendant nucleobases(naturally occurring and modified), including, but not limited to, anyof the oligomer or polymer segments referred to or claimed as peptidenucleic acids in, e.g., U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049,5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461,5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,470 6,201,103,6,228,982 and 6,357,163, WO96/04000, all of which are hereinincorporated by reference, or any of the references cited therein. Thependant nucleobase, such as, e.g., a purine or pyrimidine base on PNAmay be connected to the backbone via a linker such as, e.g., one of thelinkers taught in PCT/US02/30573 or any of the references cited therein.In one embodiment, the PNA has an N-(2-aminoethyl)-glycine) backbone.PNAs may be synthesized (and optionally labeled) as taught inPCT/US02/30573 or any of the references cited therein. PNAs hybridizetightly, and with high sequence specificity, with DNA and RNA, becausethe PNA backbone is uncharged. Thus, short PNA probes may exhibitcomparable specificity to longer DNA or RNA probes. PNA probes may alsoshow greater specificity in binding to complementary DNA or RNA.

As used herein, the term “locked nucleic acid” or “LNA” means anoligomer or polymer comprising at least one or more LNA subunits. Asused herein, the term “LNA subunit” means a ribonucleotide containing amethylene bridge that connects the 2′-oxygen of the ribose with the4′-carbon. See generally, Kurreck, Eur. J. Biochem., 270:1628-44 (2003).

Examples of nucleic acids and nucleic acid analogs also include polymersof nucleotide monomers, including double and single strandeddeoxyribonucleotides (DNA), ribonucleotides (RNA), α-anomeric formsthereof, synthetic and natural analogs thereof, and the like. Thenucleic acid chain may be composed entirely of deoxyribonucleotides,ribonucleotides, peptide nucleic acids (PNA), locked nucleic acids(LNA), synthetic or natural analogs thereof, or mixtures thereof. DNA,RNA, or other nucleic acids as defined herein can be used in the methodand compositions of the invention.

“Polar aprotic solvent” refers to an organic solvent having a dipolemoment of about 2 debye units or more, a water solubility of at leastabout 5% (volume) at or near ambient temperature, i.e., about 20° C.,and which does not undergo significant hydrogen exchange atapproximately neutral pH, i.e., in the range of 5 to 9, or in the range6 to 8. Polar aprotic solvents include those defined according to theHansen Solubility Parameters discussed below.

“Alkyldiyl” refers to a saturated or unsaturated, branched, straightchain or cyclic hydrocarbon radical having two monovalent radicalcenters derived by the removal of one hydrogen atom from each of twodifferent carbon atoms of a parent alkane, alkene, or alkyne.

“Aqueous solution” is to be understood as a solution containing water,even small amounts of water. For example, a solution containing 1% wateris to be understood as an aqueous solution.

“Hybridization application,” “hybridization assay,” “hybridizationexperiment,” “hybridization procedure,” “hybridization technique,”“hybridization method,” etc. are to be understood as referring to anyprocess that involves hybridization of nucleic acids. Unless otherwisespecified, the terms “hybridization” and “hybridization step” are to beunderstood as referring to the re-annealing step of the hybridizationprocedure as well as the denaturation step (if present).

“Hybridization composition” refers to an aqueous solution of theinvention for performing a hybridization procedure, for example, to binda probe to a nucleic acid sequence. Hybridization compositions maycomprise, e.g., at least one polar aprotic solvent, at least one nucleicacid sequence, and a hybridization solution. Hybridization compositionsdo not comprise enzymes or other components, such as deoxynucleosidetriphosphates (dNTPs), for amplifying nucleic acids in a biologicalsample.

“Hybridization solution” refers to an aqueous solution for use in ahybridization composition of the invention. Hybridization solutions arediscussed in detail below and may comprise, e.g., buffering agents,accelerating agents, chelating agents, salts, detergents, and blockingagents.

“Hansen Solubility Parameters” and “HSP” refer to the following cohesionenergy (solubility) parameters: (1) the dispersion solubility parameter(δ_(D), “D parameter”), which measures nonpolar interactions derivedfrom atomic forces; (2) the polar solubility parameter (δ_(P), “Pparameter”), which measures permanent dipole-permanent dipoleinteractions; and (3) the hydrogen bonding solubility parameter (δ_(H),“H parameter”), which measures electron exchange. The Hansen SolubilityParameters are further defined below.

“Repetitive Sequences” is to be understood as referring to the rapidlyreannealing (approximately 25%) and/or intermediately reannealing(approximately 30%) components of mammalian genomes. The rapidlyreannealing components contain small (a few nucleotides long) highlyrepetitive sequences usually found in tandem (e.g., satellite DNA),while the intermediately reannealing components contain interspersedrepetitive DNA. Interspersed repeated sequences are classified as eitherSINEs (short interspersed repeat sequences) or LINEs (long interspersedrepeated sequences), both of which are classified as retrotransposons inprimates. SINEs and LINEs include, but are not limited to, Alu-repeats,Kpn-repeats, di-nucleotide repeats, tri-nucleotide repeats,tetra-nucleotide repeats, penta-nucleotide repeats and hexa-nucleotiderepeats. Alu repeats make up the majority of human SINEs and arecharacterized by a consensus sequence of approximately 280 to 300 bpthat consist of two similar sequences arranged as a head to tail dimer.In addition to SINEs and LINEs, repeat sequences also exist inchromosome telomeres at the termini of chromosomes and chromosomecentromeres, which contain distinct repeat sequences that exist only inthe central region of a chromosome. However, unlike SINEs and LINEs,which are dispersed randomly throughout the entire genome, telomere andcentromere repeat sequences are localized within a certain region of thechromosome.

“Non-toxic” and “reduced toxicity” are defined with respect to thetoxicity labeling of formamide according to “Directive 1999/45/EC of theEuropean Parliament and of the Council of 31 May 1999 concerning theapproximation of the laws, regulations and administrative provisions ofthe Member States relating to the classification, packaging, andlabelling of dangerous preparations”(ecb.jrc.it/legislation/1999L0045EC.pdf) (“Directive”). According to theDirective, toxicity is defined using the following classification order:T+“very toxic”; T “toxic”, C “corrosive”, Xn “harmful”, .Xi “irritant.”Risk Phrases (“R phrases”) describe the risks of the classifiedtoxicity. Formamide is listed as T (toxic) and R61 (may cause harm tothe unborn child). All of the following chemicals are classified as lesstoxic than formamide: acetonitrile (Xn, R11, R20, R21, R22, R36);sulfolane (Xn, R22); γ-butyrolactone (Xn, R22, R32); and ethylenecarbonate (Xi, R36, R37, R38). At the time of filing this application,ethylene trithiocarbonate and glycol sulfite are not presently labeled.

“Denaturation” as used herein means a process in which nucleic acids orproteins reduce or lose their tertiary and/or secondary structures byapplication of compound(s), such as e.g. a strong acid or base, aconcentrated inorganic salt, an organic solvent, and/or by externalstress such as e.g. heat. This means that, when denaturation relates tonucleic acids, and when said nucleic acid is double stranded, thestrands might separate partially or completely. This further means thatthe binding interactions of the double stranded nucleic acids areweakened sufficiently by the denaturation so that hybridization withe.g. alternative complementary strands can occur more efficiently thanwithout denaturation.

“Denaturing agent” refers to any substance that is capable of loweringthe mutual binding affinity of complementary stands of nucleic acidscompared to water. Non-limiting examples of typical denaturing agentsinclude organic solvents such as formamide, urea, DMSO, andtetraalkylammonium halides or combinations thereof. Denaturationconditions are sequence dependent and are different under differentenvironmental parameters. The melting temperature (T_(m)) can be used toadjust denaturation conditions to decrease complementary base pairing inthe presence of a denaturing agent. T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. For DNA-DNA hybrids, the T_(m)can be approximated from the following equation:

T _(m)=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/L

where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs. T_(m) is reduced by about 1° C. for each 1% ofmismatching.

“Separate denaturation” as used herein, refers to hybridization methodsin which the target nucleic acid is denatured in the absence of theprobe and/or that the probe is denatured in the absence of the targetnucleic acid. For example, the target may be denatured in a firstsolution, the probe may be denatured in a second solution, and then thedenatured probe may be combined with the denatured target for a timeperiod sufficient to hybridize the target and probe. In another example,the target may be denatured in a first solution and then combined withthe probe for a time period sufficient to hybridize the target andprobe. In still a further example, the probe may be denatured in a firstsolution and then combined with the target for a time period sufficientto hybridize the target and probe.

B. Solvent Selection

Suitable polar aprotic solvents for use in the invention may be selectedbased on their Hansen Solubility Parameters. Methods for experimentallydetermining and/or calculating HSP for a solvent are known in the art,and HSP have been reported for over 1200 chemicals.

For example, the D parameter may be calculated with reasonable accuracybased on refractive index, or may be derived from charts by comparisonwith known solvents of similar size, shape, and composition afterestablishing a critical temperature and molar volume. The P parametermay be estimated from known dipole moments (see, e.g., McClellan A. L.,Tables of Experimental Dipole Moments (W.H. Freeman 1963)) usingEquation 1:

δ_(P)=37.4(Dipole Moment)/V ^(1/2)  Equation 1

where V is the molar volume. There are no equations for calculating theH parameter. Instead, the H parameter is usually determined based ongroup contributions.

HSP characterizations are conveniently visualized using a sphericalrepresentation, with the HSP of an experimentally-determined suitablereference solvent at the center of the sphere. The radius of the sphere(R) indicates the maximum tolerable variation from the HSP of thereference solvent that still allows for a “good” interaction to takeplace. Good solvents are within the sphere and bad ones are outside. Thedistance, R_(a), between two solvents based on their respective HSPvalues can be determined using Equation 2:

(R _(a))²=4(δ_(D1)−δ_(D2))²+(δ_(P1)−δ_(P2))²(δ_(H1)−δ_(H2))²  Equation 2

where subscript 1 indicates the reference sample, subscript 2 indicatesthe test chemical, and all values are in MPa^(1/2). Good solubilityrequires that R_(a) be less than the experimentally-determined radius ofthe solubility sphere R_(o). The relative energy difference between twosolvents, i.e., RED number, can be calculated by taking the ratio ofR_(a) to R_(o), as shown in Equation 3.

RED=R _(a) /R _(o)  Equation 3

RED numbers less than 1.0 indicate high affinity; RED numbers equal orclose to 1.0 indicate boundary conditions; and progressively higher REDnumbers indicate progressively lower affinities.

In some embodiments, the D parameters of the polar aprotic solvents ofthe invention are between 17.7 to 22.0 MPa^(1/2). Such relatively high Dparameters are generally associated with solvents having cyclicstructures and/or structures with sulfur or halogens. Linear compoundsare not likely to be among the most suitable polar aprotic solvents foruse in the invention, but may be considered if their P and H parametersare within the ranges discussed below. Since the D parameter ismultiplied by 4 in Equation 2, the limits are one-half of R_(o). Inaddition, it should be noted that D values of around 21 or higher areoften characteristic of a solid.

In some embodiments, the P parameters of the polar aprotic solvents ofthe invention are between 13 to 23 MPa^(1/2). Such exceptionally high Pparameters are generally associated with solvents having a high dipolemoment and presumably also a relatively low molecular volume. Forexample, for V near 60 cc/mole, the dipole moment should be between 4.5and 3.1. For V near 90 cc/mole, the dipole moment should be between 5.6and 3.9.

In some embodiments, the H parameters of the polar aprotic solvents ofthe invention are between 3 to 13 MPa^(1/2). Generally, polar aproticsolvents having an alcohol group are not useful in the compositions andmethods of the invention, since the H parameters of such solvents wouldbe too high.

The molar volume of the polar aprotic solvent may also be relevant,since it enters into the evaluation of all three Hansen SolubilityParameters. As molar volume gets smaller, liquids tend to evaporaterapidly. As molar volume gets larger, liquids tend to enter the solidregion in the range of D and P parameters recited above. Thus, the polaraprotic solvents of the invention are rather close to the liquid/solidboundary in HSP space.

In some embodiments, the polar aprotic solvents of the invention havelactone, sulfone, nitrile, sulfite, and/or carbonate functionality. Suchcompounds are distinguished by their relatively high dielectricconstants, high dipole moments, and solubility in water. An exemplarypolar aprotic solvent with lactone functionality is γ-butyrolactone(GBL), an exemplary polar aprotic solvent with sulfone functionality issulfolane (SL; tetramethylene sulfide-dioxide), an exemplary polaraprotic solvent with nitrile functionality is acetonitrile (AN), anexemplary polar aprotic solvent with sulfite functionality is glycolsulfite/ethylene sulfite (GS), and an exemplary polar aprotic solventswith carbonate functionality are ethylene carbonate (EC), propylenecarbonate (PC), or ethylene trithiocarbonate (ETC). The structures ofthese exemplary solvents are provided below and their Hansen SolubilityParameters, RED numbers, and molar volumes are given in Table 1.

TABLE 1 Molar Volume D P H RED (cm³/mole) Correlation 19.57 19.11 7.71 —— (R₀ = 3.9) GBL 19.0 16.6 7.4 0.712 76.5 PC 20.0 18.0 4.1 0.993 85.2 SL20.3 18.2 10.9 0.929 95.7 EC 19.4 21.7 5.1 0.946 66.0 ETC n/a n/a n/an/a n/a GS 20.0 15.9 5.1 n/a 75.1 n/a = not available.

Other suitable polar aprotic solvents that may be used in the inventionare cyclic compounds such as, e.g., ε-caprolactone. In addition,substituted pyrolidinones and related structures with nitrogen in a 5-or 6-membered ring, and cyclic structures with two nitrile groups, orone bromine and one nitrile group, may also be suitable for use in theinvention. For example, N-methyl pyrrolidinone (shown below) may be asuitable polar aprotic solvent for use in the methods and compositionsof the invention.

Other suitable polar aprotic solvents may contain a ring urethane group(NHCOO—). However, not all such compounds are suitable. One of skill inthe art may screen for compounds useful in the compositions and methodsof the invention as described herein. Exemplary chemicals that may besuitable for use in the invention are set forth in Tables 2 and 3 below.

TABLE 2 Solvent D P H Acetanilide 20.6 13.3 12.4 N-Acetyl Pyrrolidone17.8 13.1 8.3 4-Amino Pyridine 20.4 16.1 12.9 Benzamide 21.2 14.7 11.2Benzimidazole 20.6 14.9 11.0 1,2,3-Benzotriazole 18.7 15.6 12.4Butadienedioxide 18.3 14.4 6.2 2,3-Butylene Carbonate 18.0 16.8 3.1Caprolactone (Epsilon) 19.7 15.0 7.4 Chloro Maleic Anhydride 20.4 17.311.5 2-Chlorocyclohexanone 18.5 13.0 5.1 Chloronitromethane 17.4 13.55.5 Citraconic Anhydride 19.2 17.0 11.2 Crotonlactone 19.0 19.8 9.6Cyclopropylnitrile 18.6 16.2 5.7 Dimethyl Sulfate 17.7 17.0 9.7 DimethylSulfone 19.0 19.4 12.3 Dimethyl Sulfoxide 18.4 16.4 10.21,2-Dinitrobenzene 20.6 22.7 5.4 2,4-Dinitrotoluene 20.0 13.1 4.9Dipheynyl Sulfone 21.1 14.4 3.4 1,2-Dinitrobenzene 20.6 22.7 5.42,4-Dinitrotoluene 20.0 13.1 4.9 Epsilon-Caprolactam 19.4 13.8 3.9Ethanesulfonylchloride 17.7 14.9 6.8 Furfural 18.6 14.9 5.12-Furonitrile 18.4 15.0 8.2 Isoxazole 18.8 13.4 11.2 Maleic Anhydride20.2 18.1 12.6 Malononitrile 17.7 18.4 6.7 4-Methoxy Benzonitrile 19.416.7 5.4 1 -Methoxy-2-Nitrobenzene 19.6 16.3 5.5 1-Methyl Imidazole 19.715.6 11.2 3-Methyl Isoxazole 19.4 14.8 11.8 N-Methyl Morpholine-N- 19.016.1 10.2 Oxide Methyl Phenyl Sulfone 20.0 16.9 7.8 Methyl Sulfolane19.4 17.4 5.3 Methyl-4-Toluenesulfonate 19.6 15.3 3.8 3-Nitroaniline21.2 18.7 10.3 2-Nitrothiophene 19.7 16.2 8.2 9,10-Phenanthrenequinone20.3 17.1 4.8 Phthalic Anhydride 20.6 20.1 10.1 1,3-Propane Sultone 18.416.0 9.0 beta-Propiolactone 19.7 18.2 10.3 2-Pyrrolidone 19.4 17.4 11.3Saccharin 21.0 13.9 8.8 Succinonitrile 17.9 16.2 7.9 Sulfanilamide 20.019.5 10.7 Sulfolane 20.3 18.2 10.9 2,2,6,6- 19.5 14.0 6.3Tetrachlorocyclohexanone Thiazole 20.5 18.8 10.8 3,3,3-Trichloro Propene17.7 15.5 3.4 1,1,2-Trichloro Propene 17.7 15.7 3.4 1,2,3-TrichloroPropene 17.8 15.7 3.4

Table 2 sets forth an exemplary list of potential chemicals for use inthe compositions and methods of the invention based on their HansenSolubility Parameters. Other compounds, may of course, also meet theserequirements such as, for example, those set forth in Table 3.

TABLE 3 Chemical (dipole moment) RED Melting Point ° C. Chloroethylenecarbonate (4.02) 0.92 — 2-Oxazolidinone (5.07) 0.48 86-89 2-Imidazole1.49 90-91 1,5-Dimethyl Tetrazole (5.3) ~1.5 70-72 N-Ethyl Tetrazole(5.46) ~1.5 Trimethylene sulfide-dioxide (4.49) — — Trimethylene sulfite(3.63) — — 1,3-Dimethyl-5-Tetrazole (4.02) — — Pyridazine (3.97) 1.16 −82-Thiouracil (4.21) — — N-Methyl Imidazole (6.2) 1.28 —1-Nitroso-2-pyrolidinone −1.37 —

Some of the chemicals listed in Tables 2 and 3 have been used inhybridization and/or PCR applications in the prior art (e.g., dimethylsulfoxide (DMSO) has been used in hybridization and PCR applications,and sulfolane (SL), acetonitrile (AN), 2-pyrrolidone, ε-caprolactam, andethylene glycol have been used in PCR applications). Thus, in someembodiments, the polar aprotic solvent is not DMSO, sulfolane,acetonitrile, 2-pyrrolidone, ε-caprolactam, or ethylene glycol. However,most polar aprotic solvents have not been used in prior arthybridization applications. Moreover, even when such compounds wereused, the prior art did not recognize that they may be advantageouslyused to separately denature the probe and target in such hybridizationapplications, as disclosed in this application.

In addition, not all of the chemicals listed in Tables 2 and 3 aresuitable for use in the compositions and methods of the invention. Forexample, although DMSO is listed in Table 2 because its HansenSolubility Parameters (HSPs) fall within the ranges recited above, DMSOdoes not function to allow separate denaturation of the probe and targetin the compositions and methods of the invention. However, it is wellwithin the skill of the ordinary artisan to screen for suitablecompounds using the guidance provided herein including testing acompound in one of the examples provided. For example, in someembodiments, suitable polar aprotic solvents will have HSPs within theranges recited above and a structure shown in Formulas 1-9 above.

C. Compositions, Buffers, and Solutions

(1) Denaturation Solutions

Traditional compositions for separately or co-denaturing a probe andtarget in hybridization applications are known in the art. Suchcompositions may comprise, for example, buffering agents, acceleratingagents, chelating agents, salts, detergents, and blocking agents.

For example, the buffering agents may include SSC, HEPES, SSPE, PIPES,TMAC, TRIS, SET, citric acid, a phosphate buffer, such as, e.g.,potassium phosphate or sodium pyrrophosphate, etc. The buffering agentsmay be present at concentrations from 0.01× to 50×, such as, forexample, 0.01×, 0.1×, 0.5×, 1×, 2×, 5×, 10×, 15×, 20×, 25×, 30×, 35×,40×, 45×, or 50×. Typically, the buffering agents are present atconcentrations from 0.1× to 10×.

The accelerating agents may include polymers such as FICOLL, PVP,heparin, dextran sulfate, proteins such as BSA, glycols such as ethyleneglycol, glycerol, 1,3 propanediol, propylene glycol, or diethyleneglycol, combinations thereof such as Dernhardt's solution and BLOTTO,and organic solvents such as formamide, dimethylformamide, DMSO, etc.The accelerating agent may be present at concentrations from 1% to 80%or 0.1× to 10×, such as, for example, 0.1% (or 0.1×), 0.2% (or 0.2×),0.5% (or 0.5×), 1% (or 1×), 2% (or 2×), 5% (or 5×), 10% (or 10×), 15%(or 15×), 20% (or 20×), 25% (or 25×), 30% (or 30×), 40% (or 40×), 50%(or 50×), 60% (or 60×), 70% (or 70×), or 80% (or 80×). Typically,formamide is present at concentrations from 25% to 75%, such as 25%,30%, 40%, 50%, 60%, 70%, or 75%, while DMSO, dextran sulfate, and glycolare present at concentrations from 5% to 10%, such as 5%, 6%, 7%, 8%,9%, or 10%.

The chelating agents may include EDTA, EGTA, etc. The chelating agentsmay be present at concentrations from 0.1 mM to 10 mM, such as 0.1 mM,0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or10 mM. Typically, the chelating agents are present at concentrationsfrom 0.5 mM to 5 mM, such as 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM,3.5 mM, 4 mM, 4.5 mM, or 5 mM.

The salts may include sodium chloride, sodium phosphate, magnesiumphosphate, etc. The salts may be present at concentrations from 1 mM to750 mM, such as 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM,200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, or 750 mM. Typically,the salts are present at concentrations from 10 mM to 500 mM, such as 10mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500mM.

The detergents may include Tween, SDS, Triton, CHAPS, deoxycholic acid,etc. The detergent may be present at concentrations from 0.001% to 10%,such as, for example, 0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10%. Typically, the detergents are present at concentrations from0.01% to 1%, such as 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.

The nucleic acid blocking agents may include, for example, yeast tRNA,homopolymer DNA, denatured salmon sperm DNA, herring sperm DNA, totalhuman DNA, COT1 DNA, etc. The blocking nucleic acids may be present atconcentrations of 0.05 mg/mL to 100 mg/mL. However, the compositions andmethods of the invention surprisingly show significantly reducedbackground levels without the need for blocking agents.

A great variation exists in the literature regarding traditionaldenaturation buffers for hybridization applications. For example, atraditional solution may comprise 5× or 6×SSC, 0.01 M EDTA, 5×Dernhardt's solution, 0.5% SDS, and 100 mg/mL sheared, denatured salmonsperm DNA. Another traditional solution may comprise 50 mM HEPES, 0.5 MNaCl, and 0.2 mM EDTA. A typical solution for FISH on biologicalspecimens for RNA detection may comprise, e.g., 2×SSC, 10% dextransulfate, 2 mM vanadyl-ribonucleoside complex, 50% formamide, 0.02%RNAse-free BSA, and 1 mg/mL E. coli tRNA. A typical solution for FISH onbiological specimens for DNA detection may comprise, e.g., 2×SSC, 10%dextran sulfate, 50% formamide, and e.g., 0.3 mg/mL salmon sperm DNA or0.1 mg/mL COT1 DNA. Other typical solutions may comprise 40% formamide,10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, Alu-PNA(blocking PNA) or COT-1 DNA, and in some cases 0.1 μg/μL total human DNA(THD). Additional denaturation buffers are discussed below in thesection titled “Hybridization Conditions.”

The compositions of the invention may comprise any of the traditionalcomponents recited above in combination with at least one polar aproticsolvent. The traditional components may be present at the sameconcentrations as used in traditional denaturing solutions, or may bepresent at higher or lower concentrations, or may be omitted completely.

For example, if the compositions of the invention comprise salts such asNaCl and/or phosphate buffer, the salts may be present at concentrationsof 0-1200 mM NaCl and/or 0-200 mM phosphate buffer. In some embodiments,the concentrations of salts may be, for example, 0 mM, 15 mM, 30 mM, 45mM, 60 mM, 75 mM, 90 mM, 105 mM, 120 mM, 135 mM, 150 mM, 165 mM, 180 mM,195 mM, 210 mM, 225 mM, 240 mM, 255 mM, 270 mM, 285 mM, or 300 mM NaCland 5 mM phosphate buffer, or 600 mM NaCl and 10 mM phosphate buffer. Inother embodiments, the concentrations of salts may be, for example, theconcentrations present in 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×,0.8×, 0.9×, 1×, 2×, 3×, 4×, 5×, 6×, 7×, or 8×SSC.

If the compositions of the invention comprise accelerating agents suchas dextran sulfate, glycol, or DMSO, the dextran sulfate may be presentat concentrations of from 5% to 40%, the glycol may be present atconcentrations of from 0.1% to 10%, and the DMSO may be from 0.1% to10%. In some embodiments, the concentration of dextran sulfate may be10% or 20% and the concentration of ethylene glycol, 1,3 propanediol, orglycerol may be 1% to 10%. In some embodiments, the concentration ofDMSO may be 1%. In some embodiments, the aqueous composition does notcomprise DMSO as an accelerating agent. In some embodiments, the aqueouscomposition does not comprise formamide as an accelerating agent, orcomprises formamide with the proviso that the composition contains lessthan 25%, or less than 10%, or less than 5%, or less than 2%, or lessthan 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, orless than 0.01%.

If the compositions of the invention comprise citric acid, theconcentrations may range from 1 mM to 100 mM and the pH may range from5.0 to 8.0. In some embodiments the concentration of citric acid may be10 mM and the pH may be 6.2.

The compositions of the invention may comprise agents that reducenon-specific binding to, for example, the cell membrane, such as salmonsperm or small amounts of total human DNA or, for example, they maycomprise blocking agents to block binding of, e.g., repeat sequences tothe target such as larger amounts of total human DNA or repeat enrichedDNA or specific blocking agents such as PNA or LNA fragments andsequences. These agents may be present at concentrations of from0.01-100 μg/μL or 0.01-100 μM. For example, in some embodiments, theseagents will be 0.1 μg/μL total human DNA, or 0.1 μg/μL non-human DNA,such as herring sperm, salmon sperm, or calf thymus DNA, or 5 μMblocking PNA. However, the compositions and methods of the inventionshow significantly reduced background levels without the need forblocking agents.

One aspect of the invention is a composition or solution for separatelydenaturing the probe and target in a hybridization application. Thecomposition for denaturing the target may comprise the same componentsas the composition for denaturing the probe, or the two compositions maycomprise different components. Compositions for use in the invention mayinclude an aqueous composition comprising at least one polar aproticsolvent in an amount effective to denature double-stranded nucleotidesequences. An amount effective to denature double-stranded nucleotidesequences is an amount that enables hybridization. For example, one wayto test for whether the amount of polar aprotic solvent is effective toenable hybridization is to determine whether the polar aprotic solvent,when used in the hybridization methods and compositions describedherein, such as example 1, yield a detectable signal and/or an amplifiednucleic acid product.

Non-limiting examples of effective amounts of polar aprotic solventsinclude, e.g., about 1% to about 95% (v/v). In some embodiments, theconcentration of polar aprotic solvent is 5% to 60% (v/v). In otherembodiments, the concentration of polar aprotic solvent is 10% to 60%(v/v). In still other embodiments, the concentration of polar aproticsolvent is 30% to 50% (v/v). Concentrations of 1% to 5%, 5% to 10%, 10%,10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, or 60% to70% (v/v) are also suitable. In some embodiments, the polar aproticsolvent will be present at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%,3%, 4%, or 5% (v/v). In other embodiments, the polar aprotic solventwill be present at a concentration of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%,10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%,16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% (v/v).

If the compositions of the invention are used in a hybridization assay,they may further comprise one or more nucleic acid probes. The probesmay be directly or indirectly labeled with detectable compounds such asenzymes, chromophores, fluorochromes, and haptens. The DNA probes may bepresent at concentrations of 0.1 to 100 ng/μL. For example, in someembodiments, the probes may be present at concentrations of 1 to 10ng/μL. The PNA probes may be present at concentrations of 0.5 to 5000nM. For example, in some embodiments, the probes may be present atconcentrations of 5 to 1000 nM.

In one embodiment, a composition of the invention comprises a mixture of40% polar aprotic solvent (v/v) (e.g., ethylene carbonate, “EC”), 10%dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, and 1-10 ng/μLprobe. Another exemplary composition of the present invention comprisesa mixture of 15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphatebuffer, and 0.1 μg/μl total human DNA. Yet another exemplary compositioncomprises 15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM citric acid pH6.2, and 0.1 μg/μL non-human DNA (e.g., herring sperm, salmon sperm, orcalf thymus) OR 0.5% formamide OR 1% glycol (e.g., ethylene glycol, 1,3propanediol, or glycerol). Yet another exemplary composition comprises15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM citric acid pH 6.2. Yetanother exemplary composition comprises 15% EC and 10 mM citric acid pH6.2.

(2) Polar Aprotic Solvent(s)

Different polar aprotic solvents may impart different properties on thecompositions of the invention. For example, the choice of polar aproticsolvent may contribute to the stability of the composition, sincecertain polar aprotic solvents may degrade over time. For example, thepolar aprotic solvent ethylene carbonate breaks down into ethyleneglycol, which is a relatively stable molecule, and carbon dioxide, whichcan interact with water to form carbonic acid, altering the acidity ofthe compositions of the invention. Without being bound by theory, it isbelieved that the change in pH upon breakdown of ethylene carbonate andDNA damage from long storage makes the compositions of the inventionless effective for hybridization. However, stability can be improved byreducing the pH of the composition, by adding citric acid as a buffer atpH 6.2 instead of the traditional phosphate buffer, which is typicallyused at about pH 7.4, and/or by adding ethylene glycol atconcentrations, e.g., between 0.1% to 10%, or between 0.5% to 5%, suchas, for example, 1%, 2%, 3%, etc. For example, with 10 mM citratebuffer, the compositions of the invention are stable at 2-8° C. forapproximately 8 months. Stability can also be improved if thecompositions are stored at low temperatures (e.g., −20° C.).

In addition, certain polar aprotic solvents may cause the compositionsof the invention to separate into multi-phase systems under certainconditions. The conditions under which multi-phase systems are obtainedmay be different for different polar aprotic solvents. Generally,however, as the concentration of polar aprotic solvent increases, thenumber of phases increases. For example, compositions comprising lowconcentrations ethylene carbonate (i.e., less than 20%) may exist as onephase, while some compositions comprising higher concentrations ofethylene carbonate may separate into two, or even three phases. Forinstance, compositions comprising 15% ethylene carbonate in 20% dextransulfate, 600 mM NaCl, and 10 mM citrate buffer exist as a single phaseat room temperature, while compositions comprising 40% ethylenecarbonate in 10% dextran sulfate, 300 mM NaCl, and 5 mM phosphate bufferconsist of a viscous lower phase (approximately 25% of the total volume)and a less viscous upper phase (approximately 75% of the total volume)at room temperature. However, compositions comprising, e.g., 40% polaraprotic solvent (e.g., 40% EC in 10 mM citrate buffer) or 50% polaraprotic solvent (e.g., 50% EC in 2×SSC) is an one phase system.

On the other hand, some polar aprotic solvents may exist in two phasesat room temperature even at low concentrations. For example, sulfolane,γ-butyrolactone, ethylene trithiocarbonate, glycol sulfite, andpropylene carbonate exist as two phases at concentrations of 10, 15, 20,or 25% (20% dextran sulfate, 600 mM NaCl, 10 mM citrate buffer) at roomtemperature. In contrast, polar aprotic solvent compositions with lowerpercentages of dextran sulfate, or with no dextran sulfate, stay in onephase at room temperature (e.g. 20% GBL in 2×SSC and 20% SL in 2×SSC).

It may also be possible to alter the number of phases by adjusting thetemperature of the compositions of the invention. Generally, astemperature increases, the number of phases decreases. For example, at2-8° C., compositions comprising 40% ethylene carbonate in 10% dextransulfate, 300 mM NaCl, and 5 mM phosphate buffer may separate into athree-phase system.

It may also be possible to alter the number of phases by adjusting theconcentration of dextran sulfate and/or salt in the composition.Generally speaking, lowering the dextran sulfate concentration(traditional concentration is 10%) and/or salt concentration may reducethe number of phases. However, depending on the particular polar aproticsolvent and its concentration in the composition, single phases may beproduced even with higher concentrations of salt and dextran sulfate.For example, a composition comprising low amounts of EC (e.g., 15%, 10%,or 5%) can work well by increasing the dextran sulfate and saltconcentrations, while still keeping a one phase system. In a particularembodiment, compositions comprising a HER2 gene DNA probe, a CEN17 PNAprobe, 15% EC, 20% dextran sulfate, 600 mM NaCl, and 10 mM phosphatebuffer are frozen at −20° C. In other embodiments, the compositions areliquid at −20° C.

Some polar aprotic solvents may allow the probes to produce strongersignals in one phase or another. For example, 40% glycol sulfiteproduces strong signals in the lower phase and no signals in the upperphase. Similarly, certain types of probes may produce stronger signalsin one phase or another. For example, PNA probes tend to show strongersignals in the lower phase than the upper phase.

Accordingly, the multiphase systems of the invention may be used toconveniently examine different aspects of a sample.

Hybridization applications may be performed with a one-phase compositionof the invention, with individual phases of the multiphase compositionsof the invention, or with mixtures of any one or more of the phases in amultiphase composition of the invention. For example, in a one phasesystem, a volume of the sample may be extracted for use in thehybridization. In a mulitphase system, one may extract a volume ofsample from the phase of interest (e.g., the upper, lower, or middlephase) to use in the hybridization. Alternatively, the phases in amultiphase system may be mixed prior to extracting a volume of the mixedsample for use in the hybridization. However, the multiphase system mayyield strong and uneven local background staining depending on thecomposition. While, the addition of low amounts of formamide will reducebackground in a one phase system, it has little effect on a multiphasesystem with high concentrations (e.g., 40%) of a polar aprotic solvent.

Because the composition used in the hybridization step may differ fromthe compositions used in the separate denaturation steps, the dextransulfate and salt concentrations of the compositions of the invention arenot critical. Indeed, compositions of the invention lacking dextransulfate, salt, and buffer produce lower background (e.g., scores thatare lower by 1 to 2) and more homogenous background in hybridizationapplications in which the probe and target are separately denatured,compared to hybridization applications in which the probe and target areco-denatured. However, compositions comprising a buffer (e.g., 40% ECplus 10 mM citrate buffer) produce slightly higher background (e.g.,scores that are higher by ½) than unbuffered compositions. In oneembodiment, compositions with EC and buffer (e.g., 15% EC plus 10 mMcitrate buffer) worked without any dextran sulfate.

Hybridization applications in which the target and probe are separatelydenatured using the compositions of the invention produce morehomogenous signal intensities and a lower more homogenous backgroundstaining than hybridization applications in which the target and probeare co-denatured using traditional buffers.

(3) Optimization for Particular Applications

The compositions of the invention can be varied in order to optimizeresults for a particular application. For example, the concentration ofpolar aprotic solvent, salt, accelerating agent, blocking agent, and/orhydrogen ions (i.e. pH) may be varied in order to improve results for aparticular application.

For example, the concentration of polar aprotic solvent may be varied inorder to improve signal intensity and background staining. Generally, asthe concentration of polar aprotic solvent increases, signal intensityincreases and background staining decreases. For example, compositionsfor denaturing the probe comprising 15% EC tend to show stronger signalsand less background than compositions comprising 5% EC. However, signalintensity may be improved for compositions having low concentrations ofpolar aprotic solvent (e.g., 0% to 20%) if the concentrations of saltand/or dextran sulfate are increased. For example, strong signals may beobserved with 5% to 10% EC when the salt concentration is raisedapproximately 3 to 4 times traditional salt concentrations (i.e.,approximately 1200 mM NaCl, 20 mM phosphate buffer; traditional saltconcentrations are about 300 mM NaCl). Likewise, as lower concentrationsof polar aprotic solvent are used, higher concentrations of dextransulfate are generally required to maintain good signal and backgroundintensity.

Accordingly, the concentrations of salt and dextran sulfate may also bevaried in order to improve signal intensity and background staining.Generally, as the concentrations of salt and dextran sulfate in thecomposition for denaturing the probe increase, the signal intensityincreases and background decreases. For example, salt concentrationsthat are approximately two to four times traditional concentrations(i.e., 300 mM NaCl 5 mM phosphate buffer) produce strong signals and lowbackground. Surprisingly, however, the compositions of the invention canbe used even in the complete absence of salt. Signal intensities can beimproved under no-salt conditions by increasing the concentrations ofaccelerating agent and/or polar aprotic solvent.

Likewise, compositions for denaturing the probe exhibit increased signalintensity as dextran sulfate concentration increases from 0% to 20%.However, good signals may even be observed at dextran sulfateconcentrations of 0%. Signal intensity may be improved under low dextransulfate conditions by increasing the polar aprotic solvent and/or saltconcentrations.

In addition, the types probes used in the compositions of the inventionmay be varied to improve results. For example, in some aspects of theinvention, combinations of DNA/DNA probes may show less background thancombinations of DNA/PNA probes in the compositions of the invention orvice versa. On the other hand, PNA probes tend to show stronger signalsthan DNA probes under low salt and/or low polar aprotic solventconcentrations. In fact, PNA probes also show signals when no polaraprotic solvent is present, whereas DNA probes show weak or no signalswithout polar aprotic solvent.

A further optimization in the present invention is to separate thedenaturation of the probe and target from each other, e.g., using aspecific denaturation buffer not containing the labeled probe todenature the target. It has been found that the use of the compositionsof the inventions for such separate denaturations decreases thebackground staining and makes the staining more homogenous bothregarding the background and signal intensities. In addition, thecompositions of the invention allow the separate denaturations to occurat a low temperatures, which is beneficial, for example, to preservesample morphology and the structure of nucleic acid sequences. Asdiscussed above, the compositions of the invention are also less toxicthan, e.g., traditional formamide denaturation buffers. It is obviousfor the one known in the art that such a denaturation composition forthe target might consist of more traditional denaturation agent such ase.g. urea, DMSO or formamide, in place of the polar aprotic solvent,while the denaturation composition for the probe may contain a polaraprotic solvent in place of a more traditional denaturation agent. Theprobe and target may then be combined in the hybridizatoin step, forexample, with the fast hybridization buffer described inPCT/IB09/005893.

D. Applications, Methods, and Uses

(1) Analytical Samples

The methods and compositions of the invention may be used fully orpartly in all types of hybridization applications comprising separatedenaturation of the target and probe in the fields of cytology,histology, or molecular biology. According to one embodiment, the firstor the second nucleic acid sequence in the methods of the invention ispresent in a biological sample. Examples of such samples include, e.g.,tissue samples, cell preparations, cell fragment preparations, andisolated or enriched cell component preparations. The sample mayoriginate from various tissues such as, e.g., breast, lung, colorectal,prostate, lung, head & neck, stomach, pancreas, esophagus, liver, andbladder, or other relevant tissues and neoplasia thereof, any cellsuspension, blood sample, fine needle aspiration, ascites fluid, sputum,peritoneum wash, lung wash, urine, feces, cell scrape, cell smear,cytospin or cytoprep cells.

The sample may be isolated and processed using standard protocols. Cellfragment preparations may, e.g., be obtained by cell homogenizing,freeze-thaw treatment or cell lysing. The isolated sample may be treatedin many different ways depending of the purpose of obtaining the sampleand depending on the routine at the site. Often the sample is treatedwith various reagents to preserve the tissue for later sample analysis,alternatively the sample may be analyzed directly. Examples of widelyused methods for preserving samples are formalin-fixed followed byparaffin-embedding and cryo-preservation.

For metaphase spreads, cell cultures are generally treated withcolcemid, or anther suitable spindle pole disrupting agent, to stop thecell cycle in metaphase. The cells are then fixed and spotted ontomicroscope slides, treated with formaldehyde, washed, and dehydrated inethanol. Probes are then added and the samples are analyzed by any ofthe techniques discussed below.

Cytology involves the examination of individual cells and/or chromosomespreads from a biological sample. Cytological examination of a samplebegins with obtaining a specimen of cells, which can typically be doneby scraping, swabbing or brushing an area, as in the case of cervicalspecimens, or by collecting body fluids, such as those obtained from thechest cavity, bladder, or spinal column, or by fine needle aspiration orfine needle biopsy, as in the case of internal tumors. In a conventionalmanual cytological preparation, the sample is transferred to a liquidsuspending material and the cells in the fluid are then transferreddirectly or by centrifugation-based processing steps onto a glassmicroscope slide for viewing. In a typical automated cytologicalpreparation, a filter assembly is placed in the liquid suspension andthe filter assembly both disperses the cells and captures the cells onthe filter. The filter is then removed and placed in contact with amicroscope slide. The cells are then fixed on the microscope slidebefore analysis by any of the techniques discussed below.

In a traditional DNA hybridization experiment using a cytologicalsample, slides containing the specimen are immersed in a formaldehydebuffer, washed, and then dehydrated in ethanol. The probes are thenadded and the specimen is covered with a coverslip. The probes andspecimen are then co-denatured at a temperature sufficient to separateany double-stranded nucleic acid in the specimen (e.g. 5 minutes at 82°C.), and then incubated at a temperature sufficient to allowhybridization (e.g., overnight at 45° C.). After hybridization, thecoverslips are removed and the specimens are subjected to ahigh-stringency wash (e.g., 10 minutes at 65° C.) followed by a seriesof low-stringency washes (e.g., 2×3 minutes at room temperature). Thesamples are then dehydrated and mounted for analysis.

In a traditional RNA hybridization experiment using cytological samples,cells are equilibrated in 40% formamide, 1×SSC, and 10 mM sodiumphosphate for 5 min, incubated at 37° C. overnight in hybridizationreactions containing 20 ng of oligonucleotide probe (e.g mix of labeled50 bp oligos), 1×SSC, 40% formamide, 10% dextran sulfate, 0.4% BSA, 20mM ribonucleotide vanadyl complex, salmon testes DNA (10 mg/ml), E. colitRNA (10 mg/ml), and 10 mM sodium phosphate. Then washed twice with4×SSCI40% formamide and again twice with 2×SSC/40% formamide, both at37° C., and then with 2×SSC three times at room temperature.Digoxigenin-labeled probes can then e.g. be detected by using amonoclonal antibody to digoxigenin conjugated to Cy3. Biotin-labeledprobes can then e.g. be detected by using streptavidin-Cy5. Detectioncan be by fluorescence or CISH.

Histology involves the examination of cells in thin slices of tissue. Toprepare a tissue sample for histological examination, pieces of thetissue are fixed in a suitable fixative, typically an aldehyde such asformaldehyde or glutaraldehyde, and then embedded in melted paraffinwax. The wax block containing the tissue sample is then cut on amicrotome to yield thin slices of paraffin containing the tissue,typically from 2 to 10 microns thick. The specimen slice is then appliedto a microscope slide, air dried, and heated to cause the specimen toadhere to the glass slide. Residual paraffin is then dissolved with asuitable solvent, typically xylene, toluene, or others. These so-calleddeparaffinizing solvents are then removed with a washing-dehydratingtype reagent prior to analysis of the sample by any of the techniquesdiscussed below. Alternatively, slices may be prepared from frozenspecimens, fixed briefly in 10% formalin or other suitable fixative, andthen infused with dehydrating reagent prior to analysis of the sample.

In a traditional DNA hybridization experiment using a histologicalsample, formalin-fixed paraffin embedded tissue specimens are cut intosections of 2-6 μm and collected on slides. The paraffin is melted(e.g., 30-60 minutes at 60° C.) and then removed (deparaffinated) bywashing with xylene (or a xylene substitute), e.g., 2×5 minutes. Thesamples are rehydrated, washed, and then pre-treated (e.g., 10 minutesat 95-100° C.). The slides are washed and then treated with pepsin oranother suitable permeabilizer, e.g., 3-15 minutes at 37° C. The slidesare washed (e.g., 2×3 minutes), dehydrated, and probe is applied. Thespecimens are covered with a coverslip, the probe and specimen areco-denatured by incubating the slide at a temperature sufficient toseparate any double-stranded nucleic acid (e.g. 5 minutes at 82° C.),followed by incubation at a temperature sufficient to allowhybridization (e.g., overnight at 45° C.). After hybridization, thecoverslips are removed and the specimens are subjected to ahigh-stringency wash (e.g., 10 minutes at 65° C.) followed by a seriesof low-stringency washes (e.g., 2×3 minutes at room temperature). Thesamples are then dehydrated and mounted for analysis.

In a traditional RNA hybridization experiment using a histologicalsample, slides with FFPE tissue sections are deparaffinized in xylenefor 2×5 min, immerged in 99% ethanol 2×3 min, in 96% ethanol 2×3 min,and then in pure water for 3 min. Slides are placed in a humiditychamber, Proteinase K is added, and slides are incubated at RT for 5min-15 min. Slides are immersed in pure water for 2×3 min, immersed in96% ethanol for 10 sec, and air-dried for 5 min. Probes are added to thetissue section and covered with coverslip. The slides are incubated at55° C. in humidity chamber for 90 min. After incubation, the slides areimmersed in a Stringent Wash solution at 55° C. for 25 min, and thenimmersed in TBS for 10 sec. The slides are incubated in a humiditychamber with antibody for 30 min. The slides are immersed in TBS for 2×3min, then in pure water for 2×1 min, and then placed in a humiditychamber. The slides are then incubated with substrate for 60 min, andimmersed in tap water for 5 min.

In a traditional northern blot procedure, the RNA target sample isdenatured for 10 minutes at 65° C. in RNA loading buffer and immediatelyplaced on ice. The gels are loaded and electrophoresed with 1×MOPSbuffer (10×MOPS contains 200 mM morpholinopropansulfonic acid, 50 mMsodium acetate, 10 mM EDTA, pH 7.0) at 25 V overnight. The gel is thenpre-equilibrated in 20×SSC for 10 min and the RNA is transferred to anylon membrane using sterile 20×SSC as transfer buffer. The nucleicacids are then fixed on the membrane using, for example, UV-crosslinking at 120 mJ or baking for 30 min at 120° C. The membrane is thenwashed in water and air dried. The membrane is placed in a sealableplastic bag and prehybridized without probe for 30 min at 68° C. Theprobe is denatured for 5 min at 100° C. and immediately placed on ice.Hybridization buffer (prewarmed to 68° C.) is added and the probe ishybridized at 68° C. overnight. The membrane is then removed from thebag and washed twice for 5 min each with shaking in a low stringencywash buffer (e.g., 2×SSC, 0.1% SDS) at room temperature. The membrane isthen washed twice for 15 min each in prewarmed high stringency washbuffer (e.g., 0.1×SSC, 0.1% SDS) at 68° C. The membrane may then bestored or immediately developed for detection.

Additional examples of traditional hybridization techniques can befound, for example, in Sambrook et al., Molecular Cloning A LaboratoryManual, 2^(nd) Ed., Cold Spring Harbor Laboratory Press, (1989) atsections 1.90-1.104, 2.108-2.117, 4.40-4.41, 7.37-7.57, 8.46-10.38,11.7-11.8, 11.12-11.19, 11.38, and 11.45-11.57; and in Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1998)at sections 2.9.1-2.9.6, 2.10.4-2.10.5, 2.10.11-2.10.16, 4.6.5-4.6.9,4.7.2-4.7.3, 4.9.7-4.9.15, 5.9.18, 6.2-6.5, 6.3, 6.4, 6.3.3-6.4.9,5.9.12-5.9.13, 7.0.9, 8.1.3, 14.3.1-14.3.4, 14.9, 15.0.3-15.0.4,15.1.1-15.1.8, and 20.1.24-20.1.25.

(2) Hybridization Techniques

The compositions and methods of the present invention can be used fullyor partly in all types of nucleic acid hybridization techniquescomprising separate denaturation of the target and probe for cytologicaland histological samples. Such techniques include, for example, in situhybridization (ISH), fluorescent in situ hybridization (FISH; includingmulti-color FISH, Fiber-FISH, etc.), chromogenic in situ hybridization(CISH), silver in situ hybridization (SISH), comparative genomehybridization (CGH), chromosome paints, and arrays in situ.

Molecular probes that are suitable for use in the hybridizations of theinvention are described, e.g., in U.S. Patent Publication No.2005/0266459, which is incorporated herein by reference. In general,probes may be prepared by chemical synthesis, PCR, or by amplifying aspecific DNA sequence by cloning, inserting the DNA into a vector, andamplifying the vector an insert in appropriate host cells. Commonly usedvectors include bacterial plasmids, cosmids, bacterial artificialchromosomes (BACs), PI diverted artificial chromosomes (PACs), or yeastartificial chromosomes (YACs). The amplified DNA is then extracted andpurified for use as a probe. Methods for preparing and/or synthesizingprobes are known in the art, e.g., as disclosed in PCT/US02/30573.

In general, the type of probe determines the type of feature one maydetect in a hybridization assay. For example, total nuclear or genomicDNA probes can be used as a species-specific probe. Chromosome paintsare collections of DNA sequences derived from a single chromosome typeand can identify that specific chromosome type in metaphase andinterphase nuclei, count the number of a certain chromosome, showtranslocations, or identify extra-chromosomal fragments of chromatin.Different chromosomal types also have unique repeated sequences that maybe targeted for probe hybridization, to detect and count specificchromosomes. Large insert probes may be used to target uniquesingle-copy sequences. With these large probes, the hybridizationefficiency is inversely proportional to the probe size. Smaller probescan also be used to detect aberrations such as deletions,amplifications, inversions, duplications, and aneuploidy. For example,differently-colored locus-specific probes can be used to detecttranslocations via split-signal in situ hybridization.

In general, the ability to discriminate between closely relatedsequences is inversely proportional to the length of the hybridizationprobe because the difference in thermal stability decreases between wildtype and mutant complexes as probe length increases. Probes of greaterthan 10 bp in length are generally required to obtain the sequencediversity necessary to correctly identify a unique organism or clinicalcondition of interest. On the other hand, sequence differences as subtleas a single base (point mutation) in very short oligomers (<10 basepairs) can be sufficient to enable the discrimination of thehybridization to complementary nucleic acid target sequences as comparedwith non-target sequences.

In one embodiment, at least one set of the in situ hybridization probesmay comprise one or more PNA probes, as defined above and as describedin U.S. Pat. No. 7,105,294, which is incorporated herein by reference.Methods for synthesizing PNA probes are described in PCT/US02/30573.Alternatively, or in addition, at least one set of the hybridizationprobes in any of the techniques discussed above may comprise one or morelocked nucleic acid (LNA) probes, as described in WO 99/14226, which isincorporated herein by reference. Due to the additional bridging bondbetween the 2′ and 4′ carbons, the LNA backbone is pre-organized forhybridization. LNA/DNA and LNA/RNA interactions are stronger than thecorresponding DNA/DNA and DNA/RNA interactions, as indicated by a highermelting temperature. Thus, the compositions and methods of theinvention, which decrease the energy required for hybridization, areparticularly useful for hybridizations with LNA probes.

In one embodiment, the probes may comprise a detectable label (amolecule that provides an analytically identifiable signal that allowsthe detection of the probe-target hybrid), as described in U.S. PatentPublication No. 2005/0266459, which is incorporated herein by reference.The probes may be labeled to make identification of the probe-targethybrid possible by use, for example, of a fluorescence or bright fieldmicroscope/scanner. In some embodiments, the probe may be labeled usingradioactive labels such as ³¹P, ³³P, or ³²S, non-radioactive labels suchas digoxigenin and biotin, or fluorescent labels. The detectable labelmay be directly attached to a probe, or indirectly attached to a probe,e.g., by using a linker. Any labeling method known to those in the art,including enzymatic and chemical processes, can be used for labelingprobes used in the methods and compositions of the invention. In otherembodiments, the probes are not labeled.

In general, in situ hybridization techniques such as CGH, FISH, CISH,and SISH, employ large, mainly unspecified, nucleic acid probes thathybridize with varying stringency to genes or gene fragments in thechromosomes of cells. Using large probes renders the in situhybridization technique very sensitive. However, the successful use oflarge genomic probes in traditional hybridization assays depends onblocking the undesired background staining derived from, e.g.,repetitive sequences that are present throughout the genome. Traditionalmethods for decreasing nonspecific probe binding include saturating thebinding sites on proteins and tissue by incubating tissue withprehybridization solutions containing ficoll, bovine serum albumin(BSA), polyvinyl pyrrolidone, and nucleic acids. Such blocking steps aretime-consuming and expensive. Advantageously, the methods andcompositions of the invention reduce and/or eliminate the need for suchblocking steps, and show significantly reduced background levels withoutthe need for blocking agents and without the need for overnighthybridization in formamide-containing buffers. However, in oneembodiment, repetitive sequences may be suppressed according to themethods known in the art, e.g., as disclosed in PCT/US02/30573.

Bound probes may be detected in cytological and histological sampleseither directly or indirectly with fluorochromes (e.g., FISH), organicchromogens (e.g., CISH), silver particles (e.g., SISH), or othermetallic particles (e.g., gold-facilitated fluorescence in situhybridization, GOLDFISH). Thus, depending on the method of detection,populations of cells obtained from a sample to be tested may bevisualized via fluorescence microscopy or conventional brightfield lightmicroscopy.

Hybridization assays on cytological and histological samples areimportant tools for determining the number, size, and/or location ofspecific DNA sequences. For example, in CGH, whole genomes are stainedand compared to normal reference genomes for the detection of regionswith aberrant copy number. Typically, DNA from subject tissue and fromnormal control tissue is labeled with different colored probes. Thepools of DNA are mixed and added to a metaphase spread of normalchromosomes (or to a microarray chip, for array- or matrix-CGH). Theratios of colors are then compared to identify regions with aberrantcopy number.

FISH is typically used when multiple color imaging is required and/orwhen the protocol calls for quantification of signals. The techniquegenerally entails preparing a cytological sample, labeling probes,denaturing target chromosomes and the probe, hybridizing the probe tothe target sequence, and detecting the signal. Typically, thehybridization reaction fluorescently stains the targeted sequences sothat their location, size, or number can be determined usingfluorescence microscopy, flow cytometry, or other suitableinstrumentation. DNA sequences ranging from whole genomes down toseveral kilobases can be studied using FISH. With enhanced fluorescencemicroscope techniques, such as, for example, deconvolution, even asingle mRNA molecule can be detected. FISH may also be used on metaphasespreads and interphase nuclei.

FISH has been used successfully for mapping repetitive and single-copyDNA sequences on metaphase chromosomes, interphase nuclei, chromatinfibers, and naked DNA molecules, and for chromosome identification andkaryotype analysis through the localization of large repeated families,typically the ribosomal DNAs and major tandem array families. One of themost important applications for FISH has been in detecting single-copyDNA sequences, in particular disease related genes in humans and othereukaryotic model species, and the detection of infections agents. FISHmay be used to detect, e.g., chromosomal aneuploidy in prenataldiagnoses, hematological cancers, and solid tumors; gene abnormalitiessuch as oncogene amplifications, gene deletions, or gene fusions;chromosomal structural abnormalities such as translocations,duplications, insertions, or inversions; contiguous gene syndromes suchas microdeletion syndrome; the genetic effects of various therapies;viral nucleic acids in somatic cells and viral integration sites inchromosomes; etc. In multi-color FISH, each chromosome is stained with aseparate color, enabling one to determine the normal chromosomes fromwhich abnormal chromosomes are derived. Such techniques includemultiplex FISH (m-FISH), spectral karyotyping (SKY), combined binaryration labeling (COBRA), color-changing karyotyping, cross-species colorbanding, high resolution multicolor banding, telomeric multiplex FISH(TM-FISH), split-signal FISH (ssFISH), and fusion-signal FISH.

CISH and SISH may be used for many of the same applications as FISH, andhave the additional advantage of allowing for analysis of the underlyingtissue morphology, for example in histopathology applications. If FISHis performed, the hybridization mixture may contain sets of distinct andbalanced pairs of probes, as described in U.S. Pat. No. 6,730,474, whichis incorporated herein by reference. For CISH, the hybridization mixturemay contain at least one set of probes configured for detection with oneor more conventional organic chromogens, and for SISH, the hybridizationmixture may contain at least one set of probes configured for detectionwith silver particles, as described in Powell R D et al.,“Metallographic in situ hybridization,” Hum. Pathol., 38:1145-59 (2007).

The compositions of the invention may also be used fully or partly inall types of molecular biology techniques involving hybridization,including blotting and probing (e.g., Southern, northern, etc.), andarrays.

(3) Hybridization Conditions

The method of the present invention involves the use of polar aproticsolvents in hybridization applications comprising separate denaturationof the target and probe. The compositions of the present invention areparticularly useful for separately denaturing the probe and sample insaid methods.

Hybridization methods using the compositions of the invention mayinvolve applying the compositions to a sample comprising a targetnucleic acid sequence, most likely in a double stranded form. Usually,in order to secure access for the probe to hybridize with the targetsequence, the probe and sample are heated together to denature anydouble stranded nucleic acids. It has been argued that separatedenaturation preserves morphology better, whereas co-denaturationreduces the number of practical steps. For these reasons, separatedenaturation steps are most often used in molecular cytogeneticsapplications, and co-denaturation is most often used when tissuesections are analyzed.

Denaturation typically is performed by incubating the target and probe(either together or separately) in the presence of heat (e.g., attemperatures from about 70° C. to about 95° C.) and organic solventssuch as formamide and tetraalkylammonium halides, or combinationsthereof. For example, chromosomal DNA can be denatured by a combinationof temperatures above 70° C. (e.g., about 73° C.) and a denaturationbuffer containing 70% formamide and 2×SSC (0.3M sodium chloride and0.03M sodium citrate). Denaturation conditions typically are establishedsuch that cell morphology is preserved (e.g., relatively lowtemperatures and high formamide concentrations).

In a traditional hybridization application involving separatedenaturation of the target and probe, the target may be denatured, forexample, at 70° C. to 85° C. for 5-30 min. in buffer comprising 50% to70% formamide and 2×SSC, while the probe may be denatured, for example,at 75° C. to 95° C. for 5-10 minutes in 50% to 100% formamide. Anothertraditional protocol for separately denaturing the probe and target mayinvolve incubating the target at 75° C. for 2 min. in 70% (v/v)formamide, 10% (v/v) 20×SSC, and 10% (v/v) phosphate buffer, or in 70%(v/v) formamide, 2×SSC (pH7.0), and 0.1 mM EDTA, pH7.0, and incubatingthe probe at 75° C. for 5-10 min. in 2% (w/v) dextran sulfate, 50%formamide, 2×SSC, and 50 mM phosphate buffer. Yet another traditionalprotocol for separately denaturing the probe and target may involveincubating the target at room temperature for about 5 minutes in 0.05MNaOH and 2×SSC, and incubating the probe in 2×SSC, 50% formamide, 10%dextran sulfate, 0.15% SDS for 10 min. at 70-75° C.

In a traditional hybridization application involving co-denaturation ofthe target and probe, a typical protocol might involve incubating thetarget and probe together in 2×SSC, 50% formamide, 10% dextran sulfate,0.15% SDS for 30 sec. to 5 min. at 80° C. Another traditional protocolfor co-denaturing the target and probe may comprise incubating thetarget and probe together at 75° C. for 2-4 min. in 70% formamide and2×SSC (adjusted to pH 7.2). Yet another traditional protocol forco-denaturing the target and probe may comprise incubating the targetand probe together at 65° C. to 70° C. for 5 minutes in 50% formamide,10% dextran sulfate, and 0.1% SDS.

In the method of the invention, however, the probe and sample aredenatured in separate buffers, and then the sample and probe arecombined for the hybridization step. The sample denaturation buffer andthe probe denaturation buffer may comprise the same components, or maycomprise different components. For example, both buffers may comprise atleast one polar aprotic solvent, or only one of the two buffers maycomprise a polar aprotic solvent. The polar aprotic solvent interactswith the nucleic acids and facilitates the denaturation and re-annealingsteps. The polar aprotic solvent also allows for the use of lowerdenaturation temperatures and avoids the need for toxic chemicals, suchas formamide. As a result, the polar aprotic solvents specified in thepresent invention produce lower background and more homogenousbackground, preserve sample morphology, enable easier automation, andprovide safer (less-toxic) reagents.

Hybridizations using the denaturation compositions of the invention maybe performed using the same assay methodology as for hybridizationsperformed with traditional compositions. For example, the heatpre-treatment, digestion, denaturation, hybridization, washing, andmounting steps may use the same conditions in terms of volumes,temperatures, reagents and incubation times as for traditionalcompositions. However, the compositions of the invention provideimproved results for hybridization applications comprising separatedenaturation of the probe and sample. A great variation exists in thetraditional hybridization protocols known in the art. The compositionsof the invention may be used in any of traditional hybridizationprotocols known in the art. For example, the compositions may be used inhybridization applications comprising traditional formamidehybridization buffers, or may be used in hybridization applicationscomprising the polar aprotic solvent hybridization buffers disclosed inPCT/IB09/005893.

Alternatively, assays using the compositions of the invention can bechanged and optimized from traditional methodologies, for example, byincreasing or decreasing the temperatures and/or times used toseparately denature the target and probe. It will be understood by thoseskilled in the art that in some cases, e.g., RNA detection, denaturationsteps are not required.

For example, in some embodiments, the denaturation temperatures used toseparately denature the target and probe may vary from 55 to 100° C.,and the hybridization temperature may vary from 20 to 55° C. In otherembodiments, the denaturation temperatures may vary from 55 to 70° C.,70 to 80° C., 80 to 90° C. or 90 to 100° C., and the hybridizationtemperature may vary from 20 to 30° C., 30 to 40° C., 40 to 50° C., or50 to 55° C. In other embodiments, the denaturation temperatures may be67, 72, 82, or 92° C., and the hybridization temperature may be 37, 40,45, or 50° C.

In other embodiments, the times for separately denaturing the sample andprobe are from 0 to 15 minutes and the hybridization time may vary from0 minutes to 72 hours. In other embodiments, the denaturation times mayvary from 0 to 5 minutes, and the hybridization time may vary from 0minute to 8 hours. In other embodiments, the denaturation times may be0, 1, 2, 3, 4, 5, 10, 15, or 30 minutes, and the hybridization time maybe 0 minutes, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 180minutes, or 240 minutes.

Accordingly, hybridizations using the compositions of the invention maybe performed in less than 8 hours. In other embodiments, thehybridization step is performed in less than 6 hours. In still otherembodiments, the hybridization step is performed within 4 hours. Inother embodiments, the hybridization step is performed within 3 hours.In yet other embodiments, the hybridization step is performed within 2hours. In other embodiments, the hybridization step is performed within1 hour. In still other embodiments, the hybridization step is performedwithin 30 minutes. In other embodiments, they hybridization step cantake place within 15 minutes. The hybridization step can even take placewithin 10 minutes or in less than 5 minutes.

As hybridization time changes, the concentration of probe may also bevaried in order to produce strong signals and/or reduce background. Forexample, as hybridization time decreases, the amount of probe may beincreased in order to improve signal intensity. On the other hand, ashybridization time decreases, the amount of probe may be decreased inorder to improve background staining.

The compositions of the invention also eliminate the need for a blockingstep during hybridization applications by improving signal andbackground intensity by blocking the binding of, e.g., repetitivesequences to the target DNA. Thus, there is no need to use total humanDNA, blocking-PNA, COT-1 DNA, RNA, or DNA from any other source as ablocking agent. In addition, the compositions and methods of theinvention surprisingly show significantly reduced background levelswithout the need for overnight hybridization in formamide-containingbuffers.

The aqueous compositions of the invention furthermore provide for thepossibility to considerably reduce the concentration of nucleic acidsequences included in the composition. Generally, the concentration ofprobes may be reduced from 2 to 8-fold compared to traditionalconcentrations. For example, if HER2 DNA probes and CEN17 PNA probes areused in the compositions of the invention, their concentrations may bereduced by ¼ and ½, respectively, compared to their concentrations intraditional hybridization compositions. This feature, along with theabsence of any requirement for blocking DNA, such as blocking-PNA orCOT1, allows for an increased probe volume in automated instrumentsystems compared to the traditional 10 μL volume used in traditionalcompositions systems, which reduces loss due to evaporation, asdiscussed in more detail below.

Reducing probe concentration also reduces background. However, reducingthe probe concentration is inversely related to the hybridization time,i.e., the lower the concentration, the higher hybridization timerequired. Nevertheless, even when extremely low concentrations of probeare used with the aqueous compositions of the invention, thehybridization time is still shorter than with traditional compositions.

The compositions of the invention often allow for better signal-to-noiseratios than traditional hybridization compositions. For example, withcertain probes, a one hour hybridization with the compositions of theinvention will produce similar or lower background and stronger signalsthan an overnight hybridization in a traditional compositions.Background is not seen when no probe is added.

Those skilled in the art will understand that different type ofhybridization assays, different types of samples, different types ofprobe targets, different lengths of probes, different types of probes,e.g. DNA/RNA/PNA/LNA oligos, short DNA/RNA probes (0.5-3 kb), chromosomepaint probes, CGH, repetitive probes (e.g. alpha-satellite repeats),single-locus etc., will effect the concentrations of, e.g., salt andpolar aprotic solvents required to obtain the most effectivehybridizations. The temperatures and incubation times are also importantvariables for hybridization applications. In view of the guidanceprovided herein, one skilled in the art will understand how to varythese factors to optimize the methods of the invention.

Traditional assay methods may also be changed and optimized when usingthe compositions of the invention depending on whether the system ismanual, semi-automated, or fully automated. For example, by separatingthe denaturation of probe and target, it is possible to use a smallervolume of hybridization buffer in a more simple automated mannercompared to co-denaturation protocols, which traditionally demandtemperature ramping. Furthermore, a semi-automated or a fully automatedsystem will benefit from the short hybridization times obtained with thecompositions of the invention. The short hybridization time may reducethe difficulties encountered when traditional compositions are used insuch systems. For example, one problem with semi-automated and fullyautomated systems is that significant evaporation of the sample canoccur during hybridization, since such systems require small samplevolumes (e.g., 10-150 μL), elevated temperatures, and extendedhybridization times (e.g., 14 hours). Thus, proportions of thecomponents in traditional hybridization compositions are fairlyinvariable. However, since the compositions of the invention allow forseparate denaturation of the target and probe, evaporation is reduced,allowing for increased flexibility in the proportions of the componentsin hybridization compositions used in semi-automated and fully automatedsystems.

For example, two automated instruments have been used to performhybridizations using the compositions of the invention in hybridizationapplications having a traditional co-denaturation step (i.e., the sampleand probe were denatured together). Compositions comprising 40% ethylenecarbonate (v/v) have been used in the apparatus disclosed in PCTapplication DK2008/000430, and compositions comprising 15% ethylenecarbonate (v/v) have been used in the HYBRIMASTER HS-300 (Aloka CO. LTD,Japan). When the compositions of the invention are used in theHYBRIMASTER HS-300, the instrument can perform rapid FISH hybridizationwith water in place of the traditional toxic formamide mix, thusimproving safety and reducing evaporation. If water wetted strips areattached to the lid of the inner part of the Aloka instrument's reactionunit (hybridization chamber), e.g., as described in U.S. patentapplication Ser. No. 11/031,514, which is incorporated herein byreference, evaporation is reduced even further. Advantageously, separatedenaturation of the target and probe would decrease evaporation andsample stress in the two examples of instruments mentioned above.

Other problems with automated imaging analysis are the number of imagesneeded, the huge amount of storage place required, and the time requiredto take the images. The compositions of the invention address thisproblem by producing very strong signals compared to traditionalcompositions. Because of the very strong signals produced by thecompositions of the invention, the imaging can be done at lowermagnification than required for traditional compositions and can stillbe detected and analyzed, e.g., by algorithms. Since the focal planebecomes wider with lower magnification, the compositions of theinvention reduce or eliminate the requirement to take serial sections ofa sample. The more homogenous background staining and signal intensitiesprovided by the separate denaturation methods of the invention will alsobe beneficial for imaging analysis. As a result, the overall imaging ismuch faster, since the compositions of the invention require fewer or noserial sections and each image covers much greater area. In addition,the overall time for analysis is faster, since the total image files aremuch smaller.

Thus, the compositions and methods of the invention solve many of theproblems associated with traditional hybridization compositions andmethods.

The disclosure may be understood more clearly with the aid of thenon-limiting examples that follow, which constitute preferredembodiments of the compositions according to the disclosure. Other thanin the examples, or where otherwise indicated, all numbers expressingquantities of ingredients, reaction conditions, and so forth used in thespecification and claims are to be understood as being modified in allinstances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained herein. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should be construed in light of the number of significantdigits and ordinary rounding approaches.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope are approximations, the numerical values set forth inthe specific example are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in its respective testingmeasurements. The examples that follow illustrate the present inventionand should not in any way be considered as limiting the invention.

EXAMPLES

Reference will now be made in detail to specific embodiments of theinvention. While the invention will be described in conjunction withthese embodiments, it will be understood that they are not intended tolimit the invention to those embodiments. On the contrary, the inventionis intended to cover alternatives, modifications, and equivalents, whichmay be included within the invention as defined by the appended claims.

The reagents used in the following examples are from Dako's HistologyFISH Accessory Kit (K5599) and Cytology FISH Accessory Kit (K5499) (DakoDenmark A/S, Glostrup Denmark). The kits contain all the key reagents,except for probe, required to complete a FISH procedure forformalin-fixed, paraffin-embedded tissue section specimens. All sampleswere prepared according to the manufacturer's description. The DakoHybridizer (S2451, Dako) was used for the digestion, denaturation, andhybridization steps.

Evaluation of FISH slides was performed within a week afterhybridization using a Leica DM6000B fluorescence microscope, equippedwith DAPI, FITC, Texas Red single filters and FITC/Texas Red doublefilter under 10×, 20×, 40×, and 100× oil objective.

Evaluation of CISH slides was performed using an Olympus BX51 lightmicroscope, under 4×, 10×, 20×, 40×, and 60× objective.

In the Examples that follow, “dextran sulfate” refers to the sodium saltof dextran sulfate (D8906, Sigma) having a molecular weight M, >500,000.All concentrations of polar aprotic solvents are provided as v/vpercentages. Phosphate buffer refers to a phosphate buffered solutioncontaining NaH₂PO₄, 2H₂O (sodium phosphate dibasic dihydrate) andNa₂HPO₄, H₂O (sodium phosphate monobasic monohydrate). Citrate bufferrefers to a citrate buffered solution containing sodium citrate(Na₃C₆H₅O₇, 2H₂O; 1.06448, Merck) and citric acid monohydrate (C₆H₈O₇,H₂O; 1.00244, Merck).

General Histology FISH/CISH Procedure for Examples 1-20

The slides with cut formalin-fixed paraffin embedded (FFPE) multipletissue array sections from humans (tonsils, mammacarcinoma, kidney andcolon) were baked at 60° C. for 30-60 min, deparaffinated in xylenebaths, rehydrated in ethanol baths and then transferred to Wash Buffer.The samples were then pre-treated in Pre-Treatment Solution at a minimumof 95° C. for 10 min and washed 2×3 min. The samples were then digestedwith Pepsin RTU at 37° C. for 3 min, washed 2×3 min, dehydrated in aseries of ethanol evaporations, and air-dried. The samples were thenincubated with 10 μL FISH probe as described under the individualexperiments. The samples were then washed by Stringency Wash at 65° C.10 min, then washed 2×3 min, then dehydrated in a series of ethanolevaporations, and air-dried. Finally, the slides were mounted with 15 μLAntifade Mounting Medium. When the staining was completed, observerstrained to assess signal intensity, morphology, and background of thestained slides performed the scoring.

General Cytology FISH Procedure for Examples 21-22

Slides with metaphases preparation were fixed in 3.7% formaldehyde for 2min, washed 2×5 min, dehydrated in a series of ethanol evaporations, andair-dried. The samples were then incubated with 10 μL FISH probe asdescribed under the individual experiments. The samples were then washedby Stringency Wash at 65° C. 10 min, then washed 2×3 min, thendehydrated in a series of ethanol evaporations, and air-dried. Finally,the slides were mounted with 15 μL Antifade Mounting Medium. When thestaining was completed, observers trained to assess signal intensity andbackground of the stained slides performed the scoring as described inthe scoring for guidelines for tissue sections.

General Histology FISH/CISH Procedure for Examples 23-30

Slides with cut formalin-fixed paraffin embedded (FFPE) multiple tissuearray sections from humans (tonsils, mammacarcinoma, kidney and colon)were baked at 60° C. for 30-60 min, deparaffinated in xylene baths,rehydrated in ethanol baths and then transferred to Wash Buffer. Thesamples were then pre-treated in Pre-Treatment Solution at a minimum of95° C. for 10 min and washed 2×3 min. The samples were then digestedwith Pepsin RTU at 37° C. for 3 min, washed 2×3 min, dehydrated in aseries of ethanol evaporations, and air-dried. The samples were theneither co-denatured with 10 μL FISH probe, or the FISH probe and samplewere first separately denatured and then incubated together, asdescribed under the individual experiments. The samples were then washedin Stringency Wash buffer at 65° C. 10 min, then washed 2×3 min in WashBuffer, then dehydrated in a series of ethanol evaporations, andair-dried. Finally, the slides were mounted with 15 Antifade MountingMedium. When the staining was completed, observers trained to assesssignal intensity, morphology, and background of the stained slidesperformed the scoring.

Scoring Guidelines of Tissue Sections

The signal intensities were evaluated on a 0-3 scale with 0 meaning nosignal and 3 equating to a strong signal. The cell/tissue structures areevaluated on a 0-3 scale with 0 meaning no structure and no nucleiboundaries and 3 equating to intact structure and clear nucleiboundaries. Between 0 and 3 there are additional grades 0.5 apart fromwhich the observer can assess signal intensity, tissue structure, andbackground.

The signal intensity is scored after a graded system on a 0-3 scale.

-   -   0 No signal is seen.    -   1 The signal intensity is weak.    -   2 The signal intensity is moderate.    -   3 The signal intensity is strong.

The scoring system allows the use of ½ grades.

The tissue and nuclear structure is scored after a graded system on a0-3 scale.

-   -   0 The tissue structures and nuclear borders are completely        destroyed.    -   1 The tissue structures and/or nuclear borders are poor. This        grade includes situations where some areas have empty nuclei.    -   2 Tissue structures and/or nuclear borders are seen, but the        nuclear borders are unclear. This grade includes situations        where a few nuclei are empty.    -   3 Tissue structures and nuclear borders are intact and clear.

The scoring system allows the use of ½ grades.

The background is scored after a graded system on a 0-3 scale.

-   -   0 Little to no background is seen.    -   1 Some background.    -   2 Moderate background.    -   3 High Background.

The scoring system allows the use of ½ grades.

Example 1

This example compares the signal intensity and cell morphology fromsamples treated with the compositions of the invention or traditionalhybridization solutions as a function of denaturation temperature.

FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% formamide (15515-026, Invitrogen), 5 μM blockingPNAs (see Kirsten Vang Nielsen et al., PNA Suppression Method Combinedwith Fluorescence In Situ Hybridisation (FISH) Technique inPRINS and PNATechnologies in Chromosomal Investigation, Chapter 10 (Franck Pellestored.) (Nova Science Publishers, Inc. 2006)), 10 ng/μL Texas Red labeledCCND1 gene DNA probe (RP11-1143E20, size 192 kb).

FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate (03519, Fluka), 5 μM blockingPNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe (RP11-1143E20,size 192 kb).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were denatured as indicated for 5 min and hybridized at 45°C. for 60 minutes.

Results:

Signal Denaturation (I) (II) Cell morphology temperature Formamide ECFormamide EC 72° C. 0 2 Good Good 82° C. ½ 3 Good Good 92° C. ½ 3 Notgood Not good Signals scored as “3” were clearly visible in a 20xobjective.

Example 2

This example compares the signal intensity and background staining fromsamples treated with the compositions of the invention or traditionalhybridization solutions as a function of hybridization time.

FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% formamide, 5 μM blocking PNAs, 10 ng/μL Texas Redlabeled CCND1 gene DNA probe.

FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate, 5 μM blocking PNAs, 10 ng/μLTexas Red labeled CCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 14hours, 4 hours, 2 hours, 60 minutes, 30 minutes, 15 minutes, 0 minutes.

Results:

Signal Hybridization (I) (II) Background staining time Formamide ECFormamide EC 14 hours 3 3 +½ +2 4 hours 1 3 +½ +1 2 hours ½ 3 +0 +1 60min. ½ 3 +0 +1 30 min. 0 2½ +0 +1 15 min. 0 2 +0 +1 0 min. 0 1 +0 +½Signals scored as “3” were clearly visible in a 20x objective.

Example 3

This example compares the signal intensity from samples treated with thecompositions of the invention having different polar aprotic solvents ortraditional hybridization solutions.

FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% formamide, 5 μM blocking PNAs, 10 ng/μL Texas Redlabeled CCND1 gene DNA probe.

FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate (EC), 5 μM blocking PNAs, 10ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition III: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Propylene carbonate (PC) (540013, Aldrich), 5 μMblocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition IV: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Sulfolane (SL) (T22209, Aldrich), 5 μM blockingPNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition V: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Aceto nitrile (AN) (CO2CIIX, Lab-Scan), 5 μMblocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition VI: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% γ-butyrolactone (GBL) (B103608, Aldrich), 5 μMblocking PNAs, 7.5 ng/μL Texas Red labeled CCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

Signal (I) (II) (III) (IV) (V) (VI) Formamide EC PC SL AN GBL ½ 3 3 3 23 Signals scored as “3” were clearly visible in a 20x objective.

Example 4

This example compares the signal intensity from samples treated with thecompositions of the invention having different concentrations of polaraprotic solvent.

FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 10-60% Ethylene carbonate (as indicated), 5 μMblocking PNAs, 7.5 ng/μL Texas Red labeled IGK-constant DNA gene probe((CTD-3050E15, RP11-1083E8; size 227 kb) and 7.5 ng/μL FITClabeled/GK-variable gene DNA probe (CTD-2575M21, RP11-122B6, RP11-316G9;size 350 and 429 kb).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

Ethylene carbonate (EC) 10% 20% 30% 40% 60% Signal Texas Red 1½ 2 3 3 2intensity FITC 1 1½ 2 2½ 2 Signals scored as “3” were clearly visible ina 20x objective.

Example 5

This example compares the signal intensity and background intensity fromsamples treated with the compositions with and without PNA blocking.

FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate, 7.5 ng/μL Texas Red labeledCCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

Ethylene carbonate (EC) PNA- blocking Non- PNA blocking Signal intensity3 3 Background intensity ½+ ½+ Signals scored as “3” were clearlyvisible in a 20x objective.

Example 6

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of probe concentration andhybridization time.

FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate, and 10, 7.5, 5 or 2.5 ng/μLTexas Red labeled CCND1 gene DNA probe (as indicated).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 3hours, 2 hours and 1 hours.

Results:

Signal Intensity Hybridization (I) (II) (III) (IV) time 10 ng/μL 7.5ng/μL 5 ng/μL 2.5 ng/μL 3 hours 3 3 3 3 2 hours 3 3 3 1 1 hours 3 3 3 ½Signals scored as “3” were clearly visible in a 20x objective.

Example 7

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of salt, phosphate, andbuffer concentrations.

FISH Probe Compositions: 10% dextran sulfate, ([NaCl], [phosphatebuffer], [TRIS buffer] as indicated in Results), 40% Ethylene carbonate,7.5 ng/μL Texas Red labeled CCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

[NaCl] 300 mM 100 mM 0 mM Signal intensity 2 1 ½ phosphate [0 mM] Signalintensity 3 2½ ½ phosphate [5 mM] Signal intensity — — 3 phosphate [35mM] Signal intensity — — 2 TRIS [40 mM] Signals scored as “3” wereclearly visible in a 20x objective.

Example 8

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of dextran sulfateconcentration.

FISH Probe Compositions: 0, 1, 2, 5, or 10% dextran sulfate (asindicated), 300 mM

NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, 5 ng/μL Texas Redlabeled SIL-TAL1 gene DNA probe (RP1-278013; size 67 kb) and 6 ng/μLFITC SIL-TAL1 (ICRFc112-112C1794, RP11-184J23, RP11-8J9, CTD-2007B18,133B9; size 560 kb).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes. No blocking.

Results:

Signal Intensity % Dextran Texas Red FITC Sulfate Probe Probe 0% 1 1 1%1 1 2% 1½ 1½ 5% 2 2½ 10%  2 2½ NOTE: this experiment did not produceresults scored as “3” because the SIL-TAL1 Texas Red labeled probe isonly 67 kb and was from a non-optimized preparation.

Example 9

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of dextran sulfate, salt,phosphate, and polar aprotic solvent concentrations.

FISH Probe Composition Ia: 34% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition Ib: 34% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition Ic: 34% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 0% ethylene carbonate, 10 Texas Red labeled HER2 geneDNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition Ha: 32% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition IIb: 32% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 5% ethylene carbonate, 10 ng/g, Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition IIc: 32% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition IIIA: 30% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IIIb: 30% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition Inc: 30% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IVa: 28% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IVb: 28% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IVc: 28% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Reference V: Standard sales vial of HER2 PharmDx probe mix(K5331, Dako) containing blocking PNA. Overnight hybridization for 20hours.

All compositions were present as a single phase. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 60 minutes with noblocking, except for FISH Probe Reference V, which had PNA blocking andwas hybridized for 20 hours.

Results:

Signal Strength DNA Probes PNA Probes Composition Ia 0 ½ Composition Ib0 ½ Composition Ic ½ 2½ Composition IIa ½ 3 Composition IIb 1 2Composition IIc ½ 3 Composition IIIa 1 2½ Composition IIIb 1½ 2½Composition IIIc 2 3 Composition IVa 2½-3 3 Composition IVb 3 3Composition IVc 3 3 Reference V 2 2½ NOTE: Composition IVa gave strongDNA signals with no salt. This is not possible with standard FISHcompositions, where DNA binding is salt dependent.

Example 10

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of polar aprotic solvent anddextran sulfate concentration under high salt (4× normal) conditions.

FISH Probe Composition I: 0% ethylene carbonate, 29% dextran sulfate,1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Compositionwas a single phase.

FISH Probe Composition II: 5% ethylene carbonate, 27% dextran sulfate,1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Compositionwas a single phase.

FISH Probe Composition III: 10% ethylene carbonate, 25% dextran sulfate,1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Compositionwas a single phase.

FISH Probe Composition IV (not tested): 20% ethylene carbonate, 21%dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL TexasRed labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNAprobe. Composition had two phases.

Results:

Signal Strength DNA Probes PNA Probes Composition I ½ 3 Composition II 22½ Composition III 3 3 Composition IV — — Note: Composition II gave goodDNA signals with only 5% EC and strong DNA signals with 10% EC.

Example 11

This example compares the signal intensity and background from samplestreated with different phases of the compositions of the invention.

FISH Probe Composition: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphatebuffer, 40% Ethylene carbonate, 8 ng/μL Texas Red labeled HER2 gene DNAprobe and 600 nM FITC-labeled CEN-17 PNA probe. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 60 minutes. Noblocking.

Results:

Signal Intensity DNA Probe PNA Probe Background Upper Phase 3 1½ +2Lower Phase 3 2½ +1 Mix of Upper and 2½ 3 +½ Lower Phases NOTE: theupper phase had more background than the lower phase in theseexperiments.

Example 12

This example is similar to the previous example, but uses a differentDNA probe and GBL instead of EC.

FISH Probe Composition: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphatebuffer, 40% GBL, 10 ng/μL Texas Red labeled CCND1 gene DNA probe and 600nM FITC-labeled CEN-17 PNA probe.

The FISH probes were incubated at 82° C. for 5 min and then at 45° C.for 60 minutes. No blocking.

Results:

Signal Strength DNA Probe PNA Probe Background Top Phase 3 0-½ +1½Bottom Phase 2 ½ +3 Mixed Phases 2½ ½ +2½

Example 13

This example examines the number of phases in the compositions of theinvention as a function of polar aprotic solvent and dextran sulfateconcentration.

FISH Probe Compositions: 10 or 20% dextran sulfate; 300 mM NaCl; 5 mMphosphate buffer; 0, 5, 10, 15, 20, 25, 30% EC; 10 ng/μL probe.

Results:

Number of Phases Number of Phases % EC 10% Dextran 20% Dextran 0 1 1 5 11 10 1 1 15 1 1 20 2 2 25 2 2 30 2 2 NOTE: 15% EC, 20% dextran sulfateproduces the nicest high signal intensities of the above one phasesolution. Two phases 20% EC has even higher signal intensities than 15%.(Data not shown).

Example 14

This example compares the signal intensity and background from samplestreated with different compositions of the invention as a function ofprobe concentration and hybridization time.

FISH Probe Composition I: 10 ng/μL HER2 TxRed labeled DNA probe(standard concentration) and standard concentration of CEN7 FITC labeledPNA probe (50 nM); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer.

FISH Probe Composition II: 5 ng/μL HER2 TxRed labeled DNA probe (½ ofstandard concentration) and standard concentration (50 nM) of FITClabeled CEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer.

FISH Probe Composition III: 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ ofstandard concentration) and ½ of the standard concentration (25 nM) ofCEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer.

Compositions I-III existed as a single phase. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 3 hours, 2 hoursand 1 hours.

Results:

Hy- brid- iza- Signal Intensity tion I II III time DNA PNA B.G. DNA PNAB.G. DNA PNA B.G. 3 hours 3 3 +3 3 3 +2.5 3 3 +1.5 2 hours 2.5 2.5 +3 33 +3 3 3 +1.5 1 hours 2.5 2.5 +3 3 3 +1.5 2.5 3 +1 Signals scored as “3”were clearly visible in a 20x objective. B.G.: Back ground.

Example 15

This example compares the signal intensity and background from samplestreated with the compositions of the invention as a function of blockingagent.

FISH Probe Compositions: 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer; 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ of standardconcentration) and ½ of the standard concentration (300 nM) FITC labeledCEN17 PNA probe. Samples were blocked with: (a) nothing; (b) 0.1 μg/μLCOT1 (15279-011, Invitrogen); (c) 0.3 μg/μL COT1; or (d) 0.1 μg/μL totalhuman DNA before hybridization using the compositions of the invention.

All samples were present as a single phase. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 60 minutes.

Results:

Signal Intensity Blocking Agent Background DNA PNA Nothing +1-1.5 3 2.50.1 μg/μL COT1 +1   3 2.5 0.3 μg/μL COT1 +1.5 3 2.5 0.1 μg/μL totalhuman DNA +½ 3 2.5 NOTE: Background levels without blocking aresignificantly lower than what is normally observed by standard FISH withno blocking. In contrast, if a standard FISH composition does notcontain a blocking agent, signals normally cannot be read.

Example 16

This experiment compares different ways of removing background stainingusing the compositions of the invention.

All compositions contained 15% EC, 20% dextran sulfate, 600 mM NaCl, 10mM phosphate buffer, 2.5 ng/μL HER2 DNA probes (¼ of standardconcentration), 300 nM CEN17 PNA probe (½ of standard concentration),and one of the following background-reducing agents:

A) 5 μM blocking-PNA (see Kirsten Vang Nielsen et al., PNA SuppressionMethod Combined with Fluorescence In Situ Hybridisation (FISH) TechniqueinPRINS and PNA Technologies in Chromosomal Investigation, Chapter 10(Franck Pellestor ed.) (Nova Science Publishers, Inc. 2006))

B) 0.1 μg/μL COT-1 DNA

C) 0.1 μg/μL total human DNA (THD) (sonicated unlabelled THD)

D) 0.1 μg/tit sheared salmon sperm DNA (AM9680, Ambion)

E) 0.1 μg/μL calf thymus DNA (D8661, Sigma)

F) 0.1 μg/μL herring sperm DNA (D7290, Sigma)

G) 0.5% formamide

H) 2% formamide

I) 1% ethylene glycol (1.09621, Merck)

J) 1% glycerol (1.04095, Merck)

K) 1% 1,3-Propanediol (533734, Aldrich)

L) 1% H₂0 (control)

All samples were present as a single phase. The probes were incubated at82° C. for 5 minutes and then at 45° C. on FFPE tissue sections for 60and 120 minutes.

Results:

Signal Intensity Background blocking Hybridization/min Background DNAPNA Blocking-PNA 60 +1 3 2.5 Blocking-PNA 120 +1-1½ 3 2.5 COT-1 60 +½ 32.5 COT-1 120 +0-½  3 2.5 THD 60 +0 3 3 THD 120 +½ 3 2.5 Salmon DNAsperm 60 +0 3 3 Salmon DNA sperm 120 +0 3 3 Calf Thymus DNA 60 +0 2.5 3Calf Thymus DNA 120 +½ 3 2.5 Hearing sperm DNA 60 +0 3 3 Hearing spermDNA 120 +½ 2.5 3 0.5% formamide 60 +0 2.5 3 0.5% formamide 120 +0 3 3 2%formamide 60 +½ 2.5 3 2% formamide 120 +½ 3 3 1% Ethylene Glycol 60 +½2.5 3 1% Ethylene Glycol 120 +1½ 3 2.5 1% Glycerol 60 +½ 0.5 3 1%Glycerol 120 +1 3 2.5 1% 1,3-Propanediol 60 +0 3 2.5 1% 1,3-Propanediol120 +1 3 2.5 Nothing 60 +1 2.5 2.5 Nothing 120 +1½ 3 2.5 NOTE: allbackground reducing reagents, except for blocking-PNA, showed an effectin background reduction. Thus, specific blocking against repetitive DNAsequences is not required.

Example 17

This experiment compares the signal intensity from the upper and lowerphases using two different polar aprotic solvents.

FISH Probe Composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% ethylene trithiocarbonate (ET) (E27750, Aldrich),5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe Composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% glycol sulfite (GS) (G7208, Aldrich), 5 μMblocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

The FISH probes were incubated at 82° C. for 5 min nd then at 45° C. for60 minutes.

Results:

Signal Intensity I (ET) II (GS) Upper Phase 1½ 0 Lower Phase 0 3 Mix ofUpper and Lower Phases 2½ 3

Example 18

This experiment examines the ability of various polar aprotic solventsto form a one-phase system.

All compositions contained: 20% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, and either 10, 15, 20, or 25% of one of the followingpolar aprotic solvents:

Sulfolane

γ-Butyrolactone

Ethylene trithiocarbonate

Glycol sulfite

Propylene carbonate

Results: all of the polar aprotic solvents at all of the concentrationsexamined produced at least a two-phase system in the compositions used.However, this does not exclude that these compounds can produce aone-phase system under other composition conditions.

Example 19

This experiment examines the use of the compositions of the invention inchromogenic in situ hybridization (CISH) analysis on multi FFPE tissuesections.

FISH Probe Composition I: 4.5 ng/μL TCRAD FITC labelled gene DNA probe(¼ of standard concentration) (RP11-654A2, RP11-246A2, CTP-2355L21,RP11-158G6, RP11-780M2, RP11-481C14; size 1018 kb); 15% EC; 20% dextransulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.

FISH Probe Composition II: 4.5 ng/μL TCRAD FITC labelled gene DNA probe(¼ of standard concentration) (size 1018 kb); 15% EC; 20% dextransulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL shearedsalmon DNA sperm.

FISH Probe Composition III: 300 nM of each individual FITC labelled PNACEN17 probe (½ of standard concentration); 15% EC; 20% dextran sulfate;600 mM NaCl; 10 mM citrate buffer, pH 6.0.

All samples were analyzed using the Dako DuoCISH protocol (SK108) andcompositions for split probes with the exception that the stringencywash was conducted for 20 minutes instead of 10 minutes, and withoutusing the DuoCISH red chromogen step.

Results:

Signal Strength Composition FITC DNA FITC PNA I 3 — II 3 — III — 3 Note:The signal intensities were very strong. Due to the high levels ofbackground, it was not possible to discriminate if addition of salmonsperm DNA in Composition II reduced the background. Signals were clearlyvisible using a 10x objective in e.g. tonsils, which in general had lessbackground. If tissues possessed high background, the signals wereclearly visible using a 20x objective.

Example 20

This example compares the signal intensity and background from FFPEtissue sections treated with the compositions of the invention with twoDNA probes.

FISH Probe Composition I: 9 ng/μL IGH FITC labelled gene DNA probe(RP11-151B17, RP11-112H5, RP11-101G24, RP11-12F16, RP11-47P23,CTP-3087C18; size 612 kb); 6.4 ng/μL MYC Tx Red labeled DNA probe(CTD-2106F24, CTD-2151C21, CTD-2267H22; size 418 kb); 15% EC; 20%dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.

FISH Probe Composition II: 9 ng/μL IGH FITC labelled gene DNA probe; 6.4ng MYC TxRed labeled DNA probe; 15% EC, 20% dextran sulfate; 600 mMNaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL sheared salmon sperm DNA.

Signal Strength Salmon DNA FITC probe Texas Red probe Background − 2½ 2½+2.5 + 3 3 +1.5 NOTE: the high background was probably due to the factthat standard probe concentrations were used.

Example 21

This experiment examines the use of the compositions of the invention oncytological samples.

FISH Probe Composition: 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer; 5 ng/μL HER2 TxRed labeled DNA probe (½ of standardconcentration) and ½ of the standard concentration of CENT (25 nM).

The FISH probes were incubated on metaphase chromosome spreads at 82° C.for 5 minutes, then at 45° C. for 30 minutes, all without blocking.

Results:

Signal Strength DNA Probe PNA Probe Background 3 3 +1

No chromosome banding (R-banding pattern) was observed with thecompositions of the invention, in contrast with traditional ISIIsolutions, which typically show R-banding. A low homogenously redbackground staining of the interphase nuclei and metaphase chromosomeswas observed.

Example 22

This example compares the signal intensity and background from DNAprobes on cytology samples, metaphase spreads, with and withoutblocking.

FISH Probe Composition I: 6 ng/μL TCRAD Texas Red labelled gene DNAprobe (standard concentration) (CTP-31666K20, CTP-2373N7; size 301 kb)and 4.5 ng/μL FITC labelled gene DNA probe (¼ of standardconcentration); 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citratebuffer, pH 6.0.

FISH Probe Composition II: 6 ng/4 TCRAD Texas Red labelled gene DNAprobe (standard concentration) (size 301 kb) and 4.5 ng/μL FITC labelledgene DNA probe (¼ of standard concentration); 15% EC, 20% dextransulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL shearedsalmon sperm DNA.

The FISH probes were incubated on metaphase spreads at 82° C. for 5 min,then at 45° C. for 60 min.

Results:

Signal Intensity Blocking Agent Background Tx Red FITC Nothing +0 3 30.1 μg/μL Salmon DNA +0 3 3

Again, no chromosome banding (R-banding pattern) was observed with thecompositions of the invention. In addition, no background staining ofthe interphase nuclei or the metaphase chromosomes were observed.

Example 23

This example compares signal intensity and background for experimentsinvolving co-denaturation of the probe and specimen beforehybridization, and experiments involving separate denaturation of theprobe and specimen before hybridization.

FISH Probe Composition: 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ ofstandard concentration) and ½ of the standard concentration (300 nM) ofCEN17 PNA probes; 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mMcitrate buffer, pH 6.0.

Specimen denaturation composition: 15% EC, 20% dextran sulfate; 600 mMNaCl; 10 mM citrate buffer, pH 6.0.

Denaturation was performed as indicated in the table for 5 min.Hybridization was performed at 45° C. for 60 min. In the referencesample, the FISH probe and tissue were denatured together.

Results:

Denaturation Denaturation Temperature Temperature Signal IntensityTissue FISH probe Background DNA PNA 82° C. (reference) +2 2½ 2½ 72° C.72° C +0 3 3 82° C. 82° C +1 3 3

These results show that background staining was lower when denaturationwas performed separately on the specimen and probe. The backgroundstaining also was much more homogenous for the separate denaturationsamples (data not shown).

Example 24

This example compares signal intensity and background for experimentsinvolving co-denaturation of the probe and specimen beforehybridization, and experiments involving separate denaturation of theprobe and specimen before hybridization.

Specimen denaturation composition I: 15% EC, 10 mM citrate buffer, pH6.0 Specimen denaturation composition II: 20% EC, 10 mM citrate buffer,pH 6.0

Specimen denaturation composition III: 25% EC, 10 mM citrate buffer, pH6.0

Specimen denaturation composition IV: 30% EC, 10 mM citrate buffer, pH6.0

Specimen denaturation composition V: 40% EC, 10 mM citrate buffer, pH6.0

Specimen denaturation composition VI: 40% EC

Specimen denaturation composition VII: 15% EC, 20% dextran sulfate; 600mM NaCl; 10 mM citrate buffer, pH 6.0.

All six buffer compositions above stay in one phase at room temperature.

FISH Probe Composition: 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ ofstandard concentration) and ½ of the standard concentration (300 nM) ofCEN17 PNA probes; 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mMcitrate buffer, pH 6.0.

The probe buffer was denatured at 82° C. for 5 min. The tissue sampleswere denatured with the different Specimen denaturation compositions at82° C. for 5 min.

The slides were pre-treated as described above until the firstdehydration step. After the samples had been digested with pepsin, theslides were washed 2×3 min, and 200 μL of one of the specimendenaturation compositions was added. The slides were then covered with acoverglass and incubated on a Hybridizer (Dako) at 82° C. for 5 min. Thecoverglass was then removed, and the slides were washed 2×3 min in WashBuffer, except for the slide with Specimen denaturation composition I*,which was washed in 2×SSC. The slides were then dehydrated in 96%ethanol for 2 min. and air-dried.

The FISH probe was denatured on a heat block in a 1.5 mL centrifuge tubeat 82° C. for 5 min., and then put on ice. Ten μL of the denatured FISHprobe was added to the denatured dehydrated specimen, the slides werecoverslipped and sealed, and then hybridized at 45° C. for 60 min.Following hybridization, the specimens were treated as described above.

In the reference sample, the FISH probe and tissue were denaturedtogether.

Results:

Specimen denaturation Signal Intensity buffer Background DNA PNA I +0-½3 3 I* +0-½ 3 3 II +0-½ 3 3 III +½ 3 3 IV +½-1 3 3 V +½-1 3 3 VI +1 3 3VII +½ 3 3 Reference +2 3 3 *This slide was washed with 2x SSC for 2 × 3min., instead of Wash Buffer, after denaturation and dehydration beforeprobe application, and showed slightly increased background stainingcompared to the corresponding slide washed with Wash Buffer.

These results show that separate denaturation of the sample and theprobe significantly reduces background compared to co-denaturation ofthe probe and the specimen. The background staining also was morehomogenous than when the probe and specimen were co-denatured (data notshown).

Example 25

This example compares signal intensity and background for experimentsinvolving co-denaturation of the probe and specimen beforehybridization, and experiments involving separate denaturation of theprobe and specimen before hybridization.

FISH Probe Composition: 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ ofstandard concentration) and ½ of the standard concentration (300 nM) ofCEN17 PNA probes; 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mMcitrate buffer, pH 6.0.

Specimen denaturation composition: 15% EC, 20% dextran sulfate; 600 mMNaCl; 10 mM citrate buffer, pH 6.0.

The slides were pre-treated as described above until the firstdehydration step. After the samples had been digested with pepsin, theslides were washed 2×3 min, and 200 μL of the specimen denaturationcomposition was added. The slides were covered with a coverglass andincubated on a Hybridizer (Dako) at 72° C. for 10 min. The coverglasswas then removed, and the slides were washed 2×3 min, dehydrated in 96%ethanol for 2 min., and air-dried.

The FISH probe (aliquots of 11 μL) was denatured on a heat block in 1.5mL centrifuge tubes as indicated in the table and then put on ice. TenμL of the denatured FISH probe was added to the denatured dehydratedspecimen. The slides were coverslipped and sealed, and hybridized at 45°C. for 60 min. Following hybridization, the specimens were treated asdescribed above.

Results:

Probe Probe denaturation denaturation Signal Intensity temp timeBackground DNA PNA 62° C. 1 min +0-½ 3 3 62° C. 3 min +0-½ 3 3 62° C. 5min +0-½ 3 3 62° C. 10 min  +0-½ 3 3 72° C. 1 min +0-½ 3 3 72° C. 3 min+0-½ 3 3 72° C. 5 min +0-½ 3 3 72° C. 10 min  +0-½ 3 3

These results show that it is possible to lower the denaturationtemperature of the FISH probe to, e.g., 62° C. for 1 min. withoutnegatively impacting the signal intensity or background.

Example 26

This example compares signal intensity and background for experimentsinvolving co-denaturation of the probe and specimen beforehybridization, and experiments involving separate denaturation of theprobe and specimen before hybridization.

FISH Probe Composition I: 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ ofstandard concentration) and ½ of the standard concentration (300 nM) ofCEN17 PNA probes; 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mMcitrate buffer, pH 6.2.

Specimen denaturation composition II: 15% EC, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition III: 15% EC, 20% dextran sulfate; 600mM NaCl; 10 mM citrate buffer, pH 6.2.

The slides were pre-treated as described above until the denaturationstep. After the samples had been dehydrated, 200 μL of the specimendenaturation composition was added. The slides were covered with acoverglass and incubated on a Hybridizer (Dako) at 67° C. for 10 min.The coverglass was then removed, and the slides were washed 2×3 min,dehydrated in 96% ethanol for 2 min., and air-dried.

The FISH probe was denatured on a heat block in 1.5 mL centrifuge tubesat 67° C. for 3 min and used immediately after. Ten μL of the denaturedFISH probe was added to the denatured dehydrated specimen. The slideswere coverslipped and sealed, and hybridized at 45° C. for 60 min.Following hybridization, the specimens were treated as described above.

In the reference sample, the FISH probe and tissue were denaturedtogether at 67° C. for 10 min and hybridized at 45° C. for 60 min.

Results:

Probe Tissue Signal Intensity denaturation denaturation Background DNAPNA I II +0 3 3 I III +½-1 3 3 I (reference) +2 3 3

These results show that separate denaturation of the sample and theprobe significantly reduced background compared to co-denaturation ofthe probe and the specimen. The background staining also was morehomogenous than when the probe and specimen were co-denatured. Thebackground staining is slightly lower when the denaturation buffer doesnot contain dextran sulfate and NaCl.

Example 27

This example compares signal intensity and background for experimentsinvolving co-denaturation of the probe and specimen beforehybridization, and experiments involving separate denaturation of theprobe and specimen each with different denaturation agents beforehybridization.

FISH Probe Composition I: 40% formamide, 10% dextran sulfate, 300 mMNaCl, 5 mM phosphate buffer, 5 μM blocking PNAs, 10 ng/μL HER2 TxRedlabeled DNA gene probe standard concentration (600 mM) of CEN17 PNAprobes.

Specimen denaturation composition II: 40% formamide, 10% dextransulfate, 300 mM NaCl, 5 mM phosphate buffer, 5 μM blocking PNAs.

Specimen denaturation composition III: 15% EC, 10 mM citrate buffer, pH6.2.

The slides were pre-treated as described above until the denaturationstep. After the samples had been dehydrated, 100 μL of the specimendenaturation composition was added. The slides were covered with acoverglass and incubated on a Hybridizer (Dako) as indicated. Thecoverglass was then removed, and the slides were washed 2×3 min,dehydrated in 96% ethanol for 2 min., and air-dried.

The FISH probe was denatured on a heat block in 1.5 mL centrifuge tubesat 82° C. for 5 min and used immediately after. Ten μL of the denaturedFISH probe was added to the denatured dehydrated specimen. The slideswere coverslipped and sealed, and hybridized at 45° C. for overnight(about 20 h). Following hybridization, the specimens were treated asdescribed above.

In the reference sample, the FISH probe and tissue were denaturedtogether as indicated and hybridized at 45° C. for overnight (about 20h).

Results:

Probe Tissue denaturation Tissue denaturation Back- Signal Intensity 82°C./5 min denaturation temperature ground DNA PNA I II 67° C./10 min +½2½ 2 I II 82° C./5 min  +½ 2 2 I III 67° C./10 min +0 2½ 2½ I III 82°C./5 min  +0 2 2½ I 67° C./10 min (co-denaturation) +0 2 2½ I(reference) 82° C./5 min (co-denaturation) +0 2½ 2½

These results show that separate denaturation of the sample with ECprovides equivalent staining background and signal intensities, whencompared to co-denaturation of the probe and the specimen withformamide. The DNA signal was though slightly stronger with EC (III)than formamide (II) at the lower denaturation temperature. Thebackground staining was lower with specimen composition III (EC) thanwith specimen composition II (formamide). The best result fordenaturation at 67° C./10 min for both separate and co-denaturation wasobtained with separate denaturation using composition III (EC), whichproduced results equivalent to the reference.

Example 28

This example compares signal intensity and background for experimentsinvolving separate denaturation of the probe and specimen beforehybridization.

FISH Probe Composition I: 3.3 ng/μL HER2 TxRed labeled DNA probe (⅓ ofstandard concentration) and ½ of the standard concentration (300 nM) ofCEN17 PNA probes; 15% EC (E26258, Aldrich-Sigma), 20% dextran sulfate;600 mM NaCl; 10 mM citrate buffer, pH 6.2.

FISH Probe Composition II: 3.3 ng/μL HER2 TxRed labeled DNA probe (⅓ ofstandard concentration) and ½ of the standard concentration (300 nM) ofCEN17 PNA probes; 15% SL, 20% dextran sulfate; 600 mM NaCl; 10 mMcitrate buffer, pH 6.2.

FISH Probe Composition III: 3.3 ng/μL HER2 TxRed labeled DNA probe (⅓ ofstandard concentration) and ½ of the standard concentration (300 nM) ofCEN17 PNA probes; 15% PC, 20% dextran sulfate; 600 mM NaCl; 10 mMcitrate buffer, pH 6.2.

FISH Probe Composition IV: 3.3 ng/μL HER2 TxRed labeled DNA probe (⅓ ofstandard concentration) and ½ of the standard concentration (300 nM) ofCEN17 PNA probes; 15% GBL, 20% dextran sulfate; 600 mM NaCl; 10 mMcitrate buffer, pH 6.2.

FISH Probe Composition V: 3.3 ng/μL HER2 TxRed labeled DNA probe (⅓ ofstandard concentration) and ½ of the standard concentration (300 nM) ofCEN17 PNA probes; 15% formamide, 20% dextran sulfate; 600 mM NaCl; 10 mMcitrate buffer, pH 6.2.

Specimen denaturation composition VI: 15% EC, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition VII: 15% SL, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition VIII: 15% PC, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition IX: 15% GBL, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition X: 15% formamide, 10 mM citratebuffer, pH 6.2.

The slides were pre-treated as described above until the denaturationstep. After the samples had been dehydrated, 200 μL of the specimendenaturation composition was added. The slides were covered with acoverglass and incubated on a Hybridizer (Dako) at 67° C. for 10 min.The coverglass was then removed, and the slides were washed 2×3 min,dehydrated in 96% ethanol for 2 min., and air-dried.

The FISH probe was denatured on a heat block in tubes at 67° C. for 5min and used immediately after. Ten μL of the denatured FISH probe wasadded to the denatured dehydrated specimen. The slides were coverslippedand sealed, and hybridized at 45° C. for 60 min. Followinghybridization, the specimens were treated as described above.

Probe Tissue Signal Intensity denaturation denaturation Background DNAPNA I (EC) VI (EC) +½ 3 3 II (SL) VII (SL) +½ 1½ 2½ III (PC) VIII (PC)+2 2 3 IV (GBL) IX (GBL) +2 3 3 V (formamide) X (formamide) +½ 0 3

These results show that separate denaturation with EC, SL, PC and GBLprovide stronger signals intensities for DNA probes, compared toseparate denaturation with formamide when using short hybridizationincubation time (60 min).

Example 29

This example compares signal intensity and background for experimentsinvolving separate denaturation of the probe and specimen beforehybridization.

FISH Probe Composition I: 3.3 ng/μL HER2 TxRed labeled DNA probe (⅓ ofstandard concentration) and of the standard concentration (300 nM) ofCEN17 PNA probes; 15% EC (E26258, Aldrich-Sigma), 20% dextran sulfate;600 mM NaCl; 10 mM citrate buffer, pH 6.2.

Specimen denaturation composition II: 15% EC, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition III: 15% SL, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition IV: 15% PC, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition V: 15% GBL, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition VI: 15% formamide, 10 mM citratebuffer, pH 6.2.

The slides were pre-treated as described above until the denaturationstep. After the samples had been dehydrated, 200 μL of the specimendenaturation composition was added. The slides were covered with acoverglass and incubated on a Hybridizer (Dako) at 67° C. for 10 min.The coverglass was then removed, and the slides were washed 2×3 mM,dehydrated in 96% ethanol for 2 min., and air-dried.

The FISH probe was denatured on a heat block in tubes at 67° C. for 5min and used immediately after. Ten μL of the denatured FISH probe wasadded to the denatured dehydrated specimen. The slides were coverslippedand sealed, and hybridized at 45° C. for 60 min. Followinghybridization, the specimens were treated as described above.

Probe Tissue Signal Intensity denaturation denaturation Background DNAPNA I (EC) II (EC) +1 2½ 3 I (EC) III (SL) +1-1½ 2½-3 3 I (EC) IV (PC)+1 2½-3 3 I (EC) V (GBL) +1 3 3 I (EC) VI (formamide) +1 3 3

These results show that separate denaturation of tissue with EC (II), SL(III), PC (IV), GBL (V) and formamide (VI) provides equivalent stainingbackground and signal intensities when using a polar aprotic based probebuffer (I) and short hybridization incubation time (60 min).

Example 30

This example compares signal intensity and background for experimentsinvolving separate denaturation of the probe and specimen beforehybridization.

FISH Probe Composition I (K5331, Dako): 40% formamide, 10% dextransulfate, 300 mM NaCl, 5 mM phosphate buffer, 5 μM blocking PNAs, 10ng/μL HER2 TxRed labeled DNA gene probe standard concentration (600 mM)of CEN17 PNA probes.

Specimen denaturation composition II: 15% EC, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition III: 15% SL, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition IV: 15% PC, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition V: 15% GBL, 10 mM citrate buffer, pH6.2.

Specimen denaturation composition VI: 15% formamide, 10 mM citratebuffer, pH 6.2.

The slides were pre-treated as described above until the denaturationstep. After the samples had been dehydrated, 200 μL of the specimendenaturation composition was added. The slides were covered with acoverglass and incubated on a Hybridizer (Dako) at 67° C. for 10 min.The coverglass was then removed, and the slides were washed 2×3 min,dehydrated in 96% ethanol for 2 min., and air-dried.

The FISH probe was denatured on a heat block in tubes at 82° C. for 5min and used immediately after. Ten μL of the denatured FISH probe wasadded to the denatured dehydrated specimen. The slides were coverslippedand sealed, and hybridized at 45° C. overnight. Following hybridization,the specimens were treated as described above.

Probe Tissue Signal Intensity denaturation denaturation Background DNAPNA I (formamide) II (EC) +½ 2 3 I (formamide) III (SL) +½ 1½ 3 I(formamide) IV (PC) +½-1 3 3 I (formamide) V (GBL) +½ 2 3 I (formamide)VI (formamide) +½ 1½ 3

These results show that separate tissue denaturation with EC (II), SL(III), PC (IV) and GBL (V) provide equivalent or better signalintensities, when compared to separate tissue denaturation withformamide (VI). This was true when using a pre-denaturated traditionalformamide based probe (I) and long hybridization incubation time (about20 h). The background staining was equivalent.

FURTHER EMBODIMENTS Embodiment 1

A method of hybridizing nucleic acid sequences comprising:

-   -   combining a first nucleic acid sequence with a first aqueous        composition comprising at least one polar aprotic solvent in an        amount effective to denature a double-stranded nucleotide        sequence,    -   combining a second nucleic acid sequence with a second aqueous        composition comprising at least denaturing agent in an amount        effective to denature double-stranded nucleotide sequence, and    -   combining the first and the second nucleic acid sequence for at        least a time period sufficient to hybridize the first and second        nucleic acid sequences,    -   wherein the polar aprotic solvent is not dimethyl sulfoxide        (DMSO).

Embodiment 2

A method of hybridizing nucleic acid sequences comprising:

-   -   combining a first nucleic acid sequence with a first aqueous        composition comprising at least one polar aprotic solvent in an        amount effective to denature a double-stranded nucleotide        sequence, and    -   combining said first nucleic acid sequence with a second aqueous        composition comprising a second nucleic acid sequence and at        least one denaturing agent in an amount effective to denature        double-stranded nucleotide sequences for at least a time period        sufficient to hybridize the first and second nucleic acid        sequences,    -   wherein the polar aprotic solvent is not dimethyl sulfoxide        (DMSO).

Embodiment 3

A method of hybridizing nucleic acid sequences comprising:

-   -   combining a first nucleic acid sequence with a first aqueous        composition comprising at least one polar aprotic solvent in an        amount effective to denature a double-stranded nucleotide        sequence, and    -   combining said first nucleic acid sequence with a second nucleic        acid sequence for at least a time period sufficient to hybridize        the first and second nucleic acid sequences,

wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).

Embodiment 4

The method according to embodiments 1 or 2, wherein the denaturing agentin the second aqueous composition is a polar aprotic solvent.

Embodiment 5

The method according to any one of embodiments 1 to 4, wherein the firstnucleic acid sequence is in a biological sample.

Embodiment 6

The method according to embodiment 5, wherein the biological sample is acytology or histology sample.

Embodiment 7

The method according to any of embodiments 1-6, wherein the firstnucleic acid sequence is a single stranded sequence and the secondnucleic acid sequence is a double stranded sequence.

Embodiment 8

The method according to any of embodiments 1-6, wherein the firstnucleic acid sequence is a double stranded sequence and the secondnucleic acid sequence is a single stranded sequence.

Embodiment 9

The method according to any of embodiments 1-6, wherein the first andsecond nucleic acid sequences are double stranded sequences.

Embodiment 10

The method according to any of embodiments 1-6, wherein the first andsecond nucleic acid sequences are single stranded sequences.

Embodiment 11

The method according to any of embodiments 1-10, wherein a sufficientamount of energy to hybridize the first and second nucleic acids isprovided.

Embodiment 12

The method according to any of embodiments 1-11, wherein a sufficientamount of energy to denature the first nucleic acid is provided.

Embodiment 13

The method according to any of embodiments 1-12, wherein a sufficientamount of energy to denature the second nucleic acid is provided.

Embodiment 14

The method according to embodiments 11-13, wherein the energy isprovided by heating the compositions.

Embodiment 15

The method according to embodiment 14, wherein the heating step isperformed by the use of microwaves, hot baths, hot plates, heat wire,peltier element, induction heating or heat lamps.

Embodiment 16

The method according to any one of embodiments 12-15, wherein thetemperature for denaturing the first nucleic acid is 70° C. to 85° C.

Embodiment 17

The method according to any one of embodiments 12-16, wherein thetemperature for denaturing the second nucleic acid is 70° C. to 85° C.

Embodiment 18

The method according to any one of embodiments 12-15, wherein thetemperature for denaturing the first nucleic acid is 60° C. to 75° C.

Embodiment 19

The method according to any one of embodiments 12-15 or 18, wherein thetemperature for denaturing the second nucleic acid is 60° C. to 75° C.

Embodiment 20

The method according to any one of embodiments 12-15, wherein thetemperature for denaturing the first nucleic acid is 62° C., 67° C., 72°C., or 82° C.

Embodiment 21

The method according to any one of embodiments 12-15 or 20, wherein thetemperature for denaturing the second nucleic acid is 62° C., 67° C.,72° C., or 82° C.

Embodiment 22

The method according to any of embodiments 1-21, wherein a sufficientamount of time to denature the first nucleic acid is provided.

Embodiment 23

The method according to any of embodiments 1-22, wherein a sufficientamount of time to denature the second nucleic acid is provided.

Embodiment 24

The method according to embodiment 22 or 23, wherein the time is 1minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15minutes, or 30 minutes.

Embodiment 25

The method according to any one of embodiments 1-24, wherein the step ofhybridizing includes the steps of heating and cooling the compositions.

Embodiment 26

The method according to any one of embodiments 1-25, wherein the step ofhybridization takes less than 8 hours.

Embodiment 27

The method according to embodiment 26, wherein the step of hybridizationtakes less than 1 hour.

Embodiment 28

The method according to embodiment 27, wherein the step of hybridizationtakes less than 30 minutes.

Embodiment 29

The method according to embodiment 28, wherein the step of hybridizationtakes less than 15 minutes.

Embodiment 30

The method according to embodiment 29, wherein the step of hybridizationtakes less than 5 minutes.

Embodiment 31

The method according to any one of embodiments 1-30, further comprisinga blocking step.

Embodiment 32

The method according to any one of embodiments 1-31, wherein theconcentration of polar aprotic solvent in the aqueous composition(s) isabout 1% to 95% (v/v).

Embodiment 33

The method according to embodiment 32, wherein the concentration ofpolar aprotic solvent is 5% to 10% (v/v).

Embodiment 34

The method according to embodiment 32, wherein the concentration ofpolar aprotic solvent is 10% to 20% (v/v).

Embodiment 35

The method according to embodiment 32, wherein the concentration ofpolar aprotic solvent is 20% to 30% (v/v).

Embodiment 36

The method according to any one of embodiments 1-35, wherein the polaraprotic solvent in the aqueous composition(s) is non-toxic.

Embodiment 37

The method according to any one of embodiments 1-36, with the provisothat the aqueous composition(s) do not contain formamide.

Embodiment 38

The method according to any one of embodiments 1-36, with the provisothat the aqueous composition(s) contain less than 10% formamide.

Embodiment 39

The method according to embodiment 38, with the proviso that the aqueouscomposition(s) contain less than 2% formamide.

Embodiment 40

The method according to embodiment 39, with the proviso that the aqueouscomposition(s) contains less than 1% formamide.

Embodiment 41

The method according to any of embodiments 1-40, wherein the polaraprotic solvent in the aqueous composition(s) has lactone, sulfone,nitrile, sulfite, and/or carbonate functionality.

Embodiment 42

The method according to any one of embodiments 1-41, wherein the polaraprotic solvent in the aqueous composition(s) has a dispersionsolubility parameter between 17.7 to 22.0 MPa^(1/2), a polar solubilityparameter between 13 to 23 MPa^(1/2), and a hydrogen bonding solubilityparameter between 3 to 13 MPa^(1/2).

Embodiment 43

The method according to any one of embodiments 1-42, wherein the polaraprotic solvent in the aqueous composition(s) has a cyclic basestructure.

Embodiment 44

The method according to any one of embodiments 1-43, wherein the polaraprotic solvent in the aqueous composition(s) is selected from the groupconsisting of:

where X is O and R1 is alkyldiyl, and

where X is optional and if present, is chosen from O or S,

where Z is optional and if present, is chosen from O or S,

where A and B independently are O or N or S or part of the alkyldiyl ora primary amine,

where R is alkyldiyl, and

where Y is O or S or C.

Embodiment 45

The method according to any one of embodiments 1-44, wherein the polaraprotic solvent in the aqueous composition(s) is selected from the groupconsisting of: acetanilide, acetonitrile, N-acetyl pyrrolidone, 4-aminopyridine, benzamide, benzimidazole, 1,2,3-benzotriazole,butadienedioxide, 2,3-butylene carbonate, γ-butyrolactone, caprolactone(epsilon), chloro maleic anhydride, 2-chlorocyclohexanone,chloroethylene carbonate, chloronitromethane, citraconic anhydride,crotonlactone, 5-cyano-2-thiouracil, cyclopropylnitrile, dimethylsulfate, dimethyl sulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyltetrazole, 1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate,N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate,ethylene glycol sulfate, glycol sulfite, furfural, 2-furonitrile,2-imidazole, isatin, isoxazole, malononitrile, 4-methoxy benzonitrile,1-methoxy-2-nitrobenzene, methyl alpha bromo tetronate, 1-methylimidazole, N-methyl imidazole, 3-methyl isoxazole, N-methylmorpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone,methyl sulfolane, methyl-4-toluenesulfonate, 3-nitroaniline,nitrobenzimidazole, 2-nitrofuran, 1-nitroso-2-pyrolidinone,2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenylsydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine),1,3-propane sultone, β-propiolactone, propylene carbonate,4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone,saccharin, succinonitrile, sulfanilamide, sulfolane,2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide,tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil,3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloropropene, trimethylene sulfide-dioxide, and trimethylene sulfite.

Embodiment 46

The method according to any one of embodiments 1-44, wherein the polaraprotic solvent in the aqueous composition(s) is selected from the groupconsisting of:

Embodiment 47

The method according to any one of embodiments 1-44, wherein the polaraprotic solvent in the aqueous composition(s) is:

Embodiment 48

The method according to any one of embodiments 1-47, wherein the aqueouscomposition(s) further comprise at least one additional componentselected from the group consisting of: buffering agents, salts,accelerating agents, chelating agents, detergents, and blocking agents.

Embodiment 49

The method according to embodiment 48, wherein the accelerating agent isdextran sulfate and the salts are NaCl and/or phosphate buffer.

Embodiment 50

The method according to embodiment 49, wherein the dextran sulfate ispresent at a concentration of 5% to 40%, the NaCl is present at aconcentration of 0 mM to 1200 mM, and/or the phosphate buffer is presentat a concentration of OrnM to 50 mM.

Embodiment 51

The method according to embodiment 50, wherein the dextran sulfate ispresent at a concentration of 10% to 30%, the NaCl is present at aconcentration of 300 mM to 600 mM, and/or the phosphate buffer ispresent at a concentration of 5 mM to 20 mM.

Embodiment 52

The method according to embodiment 48, wherein the accelerating agent isselected from the group consisting of: formamide, DMSO, glycerol,propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol,glycol, and 1,3 propanediol, and the buffering agent is citric acidbuffer.

Embodiment 53

The method according to embodiment 52, wherein the formamide is presentat a concentration of 0.1-5%, the DMSO is present at a concentration of0.01% to 10%, the glycerol, propylene glycol, 1,2-propanediol,diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol arepresent at a concentration of 0.1% to 10%, and the citric acid buffer ispresent at a concentration of 1 mM to 50 mM.

Embodiment 54

The method according to embodiment 48, wherein the blocking agent isselected from the group consisting of: total human DNA, herring spermDNA, salmon sperm DNA, and calf thymus DNA.

Embodiment 55

The method according to embodiment 54, wherein the total human DNA,herring sperm DNA, salmon sperm DNA, and calf thymus DNA are present ata concentration of 0.01 to 10 μg/μL.

Embodiment 56

The method according to embodiment 48, wherein the aqueouscomposition(s) comprise 40% of at least one polar aprotic solvent, 10%dextran sulfate, 300 mM NaCl, and/or 5 mM phosphate buffer.

Embodiment 57

The method according to embodiment 48, wherein the aqueouscomposition(s) comprise 15% of at least one polar aprotic solvent, 20%dextran sulfate, 600 mM NaCl, and/or 10 mM phosphate buffer.

Embodiment 58

The method according to embodiment 48, wherein the aqueouscomposition(s) comprise 15% of at least one polar aprotic solvent, 20%dextran sulfate, 600 mM NaCl, and 10 mM citric acid buffer pH 6.2.

Embodiment 59

The method according to any one of embodiments 1-58, wherein the aqueouscomposition(s) comprise one phase at room temperature.

Embodiment 60

The method according to any one of embodiments 1-58, wherein the aqueouscomposition(s) comprise multiple phases at room temperature.

Embodiment 61

The method according to embodiment 60, wherein the aqueouscomposition(s) comprise two phases at room temperature.

Embodiment 62

The method according to embodiment 60 or 61, wherein the phases of theaqueous composition(s) are mixed.

Embodiment 63

An aqueous composition for performing separate denaturation of a targetin a hybridization application, said composition comprising at least onepolar aprotic solvent in an amount effective to denature adouble-stranded nucleotide sequence, wherein the polar aprotic solventis not dimethyl sulfoxide (DMSO).

Embodiment 64

The aqueous composition of embodiment 63, wherein the concentration ofpolar aprotic solvent is defined as in any one of embodiments 32 to 35.

Embodiment 65

The aqueous composition of embodiment 63 or 64, wherein the polaraprotic solvent is defined as in any one of embodiments 36 or 41 to 47.

Embodiment 66

The aqueous composition of any one of embodiments 61 to 65, wherein theaqueous composition is defined as in any one of embodiments 37 to 40 or48 to 62.

Embodiment 67

Use of a composition comprising between 1 and 95% (v/v) of at least onepolar aprotic solvent for performing a separate denaturation of a targetin a hybridization application.

Embodiment 68

Use of a composition according to embodiment 67, wherein theconcentration of polar aprotic solvent is defined as in any one ofembodiments 32 to 35.

Embodiment 69

Use of a composition according to embodiment 67 or 68, wherein the polaraprotic solvent is defined as in any one of embodiments 36 or 41 to 47.

Embodiment 70

Use of a composition according to any one of embodiments 67 to 69,wherein the aqueous composition is defined as in any one of embodiments37 to 40 or 48 to 62.

Embodiment 71

A kit for performing a hybridization assay comprising:

-   -   a first aqueous composition according to any one of embodiments        63-66; and    -   a second aqueous composition comprising at least one nucleic        acid sequence.

Embodiment 72

The kit according to embodiment 71, wherein the second aqueouscomposition further comprises at least one denaturing agent in an amounteffective to denature double-stranded nucleotide sequences.

Embodiment 73

The kit according to embodiment 72, wherein the denaturing agent in thesecond aqueous composition is a polar aprotic solvent.

Embodiment 74

The kit according to embodiment 73, wherein the concentration of polaraprotic solvent in the second aqueous composition is defined as in anyone of embodiments 32 to 35.

Embodiment 75

The kit according to embodiment 73 or 74, wherein the polar aproticsolvent in the second aqueous composition is defined as in any one ofembodiments 36 or 41 to 47.

Embodiment 76

The kit according to any one of embodiments 71 to 75, wherein the secondaqueous composition is defined as in any one of embodiments 37 to 40 or48 to 62.

1-76. (canceled)
 77. A method of hybridizing nucleic acid sequencescomprising: combining a first nucleic acid sequence within a sample witha first aqueous composition comprising at least one polar aproticsolvent and at least one accelerating agent, wherein the polar aproticsolvent is in an amount effective to denature a double-strandednucleotide sequence and is not dimethyl sulfoxide (DMSO); and combiningsaid first nucleic acid sequence with a second nucleic acid sequence forat least a time period sufficient to hybridize the first and secondnucleic acid sequences.
 78. The method according to claim 77, wherein(a) a sufficient amount of energy to denature the first nucleic acidsequence is provided, and/or (b) a sufficient amount of energy todenature the second nucleic acid sequence is provided, wherein theenergy is provided by heating.
 79. The method according to claim 78,wherein the heating to denature the first nucleic acid sequence and/orthe second nucleic acid sequence is at a temperature of 70° C. to 85° C.80. The method according to claim 78, wherein the heating to denaturethe first nucleic acid sequence and/or the second nucleic acid sequenceis at a temperature of 67° C. to 92° C.
 81. The method according toclaim 78, wherein the first nucleic acid sequence and/or the secondnucleic acid sequence is denatured for a time of 0 to 15 minutes. 82.The method according to claim 77, wherein the step of hybridizingincludes steps of heating and cooling the compositions, and the step ofhybridization is performed within 3 hours.
 83. The method according toclaim 77, wherein the step of hybridizing includes steps of heating andcooling the compositions, and the step of hybridization is performedwithin 2 hours.
 84. The method according to claim 77, wherein the firstaqueous composition comprises one phase at room temperature.
 85. Themethod according to claim 77, wherein the second nucleic acid sequenceis in a second aqueous composition, and the second aqueous compositioncomprises one phase at room temperature.
 86. The method according toclaim 77, wherein the first nucleic acid sequence and the second nucleicacid sequence are combined and hybridized in a volume of 10 μL to 150μL.
 87. The method according to claim 77, wherein the polar aproticsolvent has lactone, sulfone, nitrile, sulfite, and/or carbonatefunctionality.
 88. The method according to claim 77, wherein the polaraprotic solvent has a cyclic base structure.
 89. The method according toclaim 77, wherein the polar aprotic solvent in the first aqueouscomposition is selected from the group consisting of:

where X is O and R1 is alkyldiyl, and

where X is optional and if present, is chosen from O or S, where Z isoptional and if present, is chosen from O or S, where A and Bindependently are O or N or S or part of the alkyldiyl or a primaryamine, where R is alkyldiyl, and where Y is O or S or C.
 90. The methodaccording to claim 77, wherein the polar aprotic solvent has aconcentration in the first aqueous composition of about 1% to 90% (v/v).91. The method according to claim 77, wherein the polar aprotic solventhas a concentration in the first aqueous composition of 5% to 10% (v/v).92. The method according to claim 77, wherein the polar aprotic solventhas a concentration in the first aqueous composition of 10% to 20%(v/v).
 93. The method according to claim 77, wherein the polar aproticsolvent has a concentration in the first aqueous composition of 20% to30% (v/v).
 94. The method according to claim 77, wherein the polaraprotic solvent is selected from the group consisting ofγ-butyrolactone, sulfolane, glycol sulfite, ethylene carbonate,propylene carbonate, ethylene thiocarbonate, and combinations thereof.95. The method according to claim 77, wherein the first aqueouscomposition further comprise at least one additional component selectedfrom the group consisting of: buffering agents, salts, chelating agents,detergents, blocking agents, and combinations thereof.
 96. The methodaccording to claim 77, wherein the first aqueous composition furthercomprises a salt selected from sodium chloride, sodium phosphate,magnesium phosphate, and combinations thereof.
 97. The method accordingto claim 77, wherein the first aqueous composition further comprisesTris buffer.
 98. The method according to claim 77, wherein theaccelerating agent is present at a concentration of 10% to 40%.
 99. Themethod according to claim 77, wherein the accelerating agent is selectedfrom the group consisting of FICOLL, polyvinylpyrrolidone (PVP),heparin, dextran sulfate, proteins, glycols, organic solvents, andcombinations thereof.
 100. The method according to claim 77, wherein theaccelerating agent is selected from the group consisting of: FICOLL,polyvinylpyrrolidone (PVP), heparin, dextran sulfate, bovine serumalbumin (BSA), formamide, DMSO, glycerol, propylene glycol,1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and1,3-propanediol, and combinations thereof.
 101. A kit for performing ahybridization assay comprising: a first aqueous composition forperforming denaturation of a target in a hybridization application, saidfirst aqueous composition comprising at least one polar aprotic solventand an accelerating agent, wherein the polar aprotic solvent is in anamount effective to denature a double-stranded nucleotide sequence andis not dimethyl sulfoxide (DMSO); and a second aqueous compositioncomprising at least one nucleic acid sequence.
 102. The kit according toclaim 101, wherein the at least one nucleic acid sequence in the secondaqueous composition is a FISH probe.
 103. The kit according to claim101, wherein the first aqueous composition comprises one phase at roomtemperature.
 104. The kit according to claim 101, wherein the secondaqueous composition comprises one phase at room temperature.